Formulation for anti-α4β7 antibody

ABSTRACT

Antibody formulations are described comprising a mixture of a non-reducing sugar, an anti-α4β7 antibody and at least one amino acid. The disclosed formulations have improved stability, reduced aggregate formation, and may retard degradation of the anti-α4β7 antibody therein or exhibit any combinations thereof. The present invention further provides a safe dosing regimen of these antibody formulations that is easy to follow, and which results in a therapeutically effective amount of the anti-α4β7 antibody in vivo.

RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/US2012/036072, filed May 2, 2012, published in English, andclaims the benefit of U.S. Provisional Application 61/585,859, filed onJan. 12, 2012, U.S. Provisional Application 61/550,545, filed on Oct.24, 2011, and U.S. Provisional Application 61/481,533 filed on May 2,2011. The entire contents of the foregoing applications are herebyincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 30, 2012, isnamed 92596615.txt and is 17,024 bytes in size.

BACKGROUND OF THE INVENTION

Advances in biotechnology have made it possible to produce a variety ofproteins for pharmaceutical applications using recombinant DNAtechniques. Because proteins are larger and more complex thantraditional organic and inorganic drugs (i.e. possessing multiplefunctional groups in addition to complex three-dimensional structures),the formulation of such proteins poses special problems. For a proteinto remain biologically active, a formulation must preserve theconformational integrity of at least a core sequence of the protein'samino acids, while at the same time protecting the protein's multiplefunctional groups from degradation. Proteins may suffer from a lack ofstability, and monoclonal and polyclonal antibodies in particular may berelatively unstable (See e.g., Wang et al., J. Pharm Sci. 96:1-26(2007)). A large number of formulation options are available, and notone approach or system is suitable for all proteins. Several factors tobe considered have been reported (See e.g., Wang et al.).

Numerous characteristics may affect a protein's stability. In fact, evenin the case of purified antibodies, the antibody structures may beheterogeneous, which further complicates the formulation of suchsystems. Moreover, the excipients included in antibody formulationspreferably minimize any potential immune response.

In the case of antibodies, preservation of the conformational integrityis even more important. Degradation pathways for proteins can involvechemical instability (i.e., any process which involves modification ofthe protein by bond formation or cleavage resulting in a new chemicalentity) or physical instability (i.e., changes in the higher orderstructure of the protein). Chemical instability is manifested in, forexample, deamidation, isomerization, hydrolysis, oxidation,fragmentation, glycan beta elimination or disulfide exchange. Physicalinstability can result from denaturation, aggregation, precipitation oradsorption, for example. The four most common protein degradationpathways are protein fragmentation, aggregation, deamidation, andoxidation. Consequences of chemical or physical instability oftherapeutic protein include a lowering of the effective administereddose, decreased safety of the therapy due to, for example irritation orimmunological reactivity, and more frequent manufacturing due to shortshelf life.

Freeze-drying is a commonly employed technique for preserving proteins;freeze-drying serves to remove water from the protein preparation ofinterest. Freeze-drying, or lyophilization, is a process by which thematerial to be dried is first frozen and then the ice or frozen solventis removed by sublimation under vacuum. Excipients can be included inthe pre-lyophilized formulation to stabilize proteins during thelyophilization process and/or to improve the stability of thelyophilized protein formulation (Pikal M., Biopharm. 3(9)26-30 (1990)and Arakawa et al. Pharm. Res. 8(3):285-291 (1991)).

Several publications have disclosed generally various methods oftreating inflammatory bowel diseases, and provided dosing schemes foradministration of agents designed to treat inflammatory bowel disease.For example, WO 96/24673 discloses mucosal vascular addressins andtreatment of diseases associated with leukocyte recruitment to thegastrointestinal tract as a result of leukocyte binding to cellsexpressing MAdCAM. U.S. 2005/0095238 describes methods of treating adisease associated with leukocyte infiltration of mucosal tissue andadministration to a human an effective amount of a human or humanizedimmunoglobulin or antigen binding fragment having binding specificityfor α4β7 integrin. U.S. 2005/0095238 further describes various doses(e.g. 0.15, about 0.5, about 1.0, about 1.5 or about 2.0 mgimmunoglobulin or fragment per kg body weight) and various intervalsbetween doses (7, 14, 21, 28, or 30 days). However, the aforementionedpatents and publications do not disclose specific formulations of theanti-α4β7 antibody or the specific doses and dose regimens described andclaimed herein. Importantly, the aforementioned patents do not discloseformulations, doses, and dose regimens that provide for the methods oftreatment (supported by clinical trial data) described and claimedherein.

The antibody formulations of the present invention may be useful forinhibiting leukocyte binding to cells expressing MAdCAM and thereforeaid in treatment of inflammatory bowel diseases in patients. There is,accordingly, an urgent need to discover suitable dosages and dosingschedules of these compounds, and to develop formulations, preferablyintravenous formulations, which give rise to steady, therapeuticallyeffective blood levels of the antibody formulations over an extendedperiod of time in a stable and convenient form.

SUMMARY OF THE INVENTION

The invention relates to the identification of a non-reducing sugar andat least one amino acid, as useful excipients for formulating anti-α4β7antibody formulations whose instability makes them susceptible todeamidation, oxidation, isomerization and/or aggregation. Theformulation improves stability, reduces aggregate formation and retardsdegradation of the antibody therein.

Thus, in a first aspect, the invention relates to a stable formulationcomprising a mixture of a non-reducing sugar, an anti-α4β7 antibody andat least one free amino acid, and the molar ratio of non-reducing sugarto anti-α4β7 antibody (mole:mole) is greater than 600:1. The formulationmay be a liquid formulation or a dry formulation (e.g., lyophilized).The formulation can also contain a buffering agent. In some embodiments,the non-reducing sugar is mannitol, sorbitol, sucrose, trehalose, or anycombination thereof.

In some embodiments, the free amino acid of the formulation ishistidine, alanine, arginine, glycine, glutamic acid, or any combinationthereof. The formulation can comprise between about 50 mM to about 175mM of free amino acid. The formulation can comprise between about 100 mMand about 175 mM of free amino acid. The ratio of free amino acid toantibody molar ratio can be at least 250:1.

The formulation can also contain a surfactant. The surfactant can bepolysorbate 20, polysorbate 80, a poloxamer, or any combination thereof.

In some aspects, the formulation can minimize immunogenicity of theanti-α4β7 antibody.

The formulation, e.g., in the dried state, can be stable for at leastthree months at 40° C., 75% relative humidity (RH).

In another aspect, the formulation is lyophilized and comprises at leastabout 5% to about 10% anti-α4β7 antibody before lyophilization. Theformulation can contain at least about 6% anti-α4β7 antibody beforelyophilization. The formulation can be reconstituted from a lyophilizedformulation (e.g., reconstituted to comprise a stable liquidformulation).

In another aspect, the invention relates to a stable formulationcomprising a mixture of a non-reducing sugar, an anti-α4β7 antibody andat least one free amino acid, and the molar ratio of non-reducing sugarto anti-α4β7 antibody (mole:mole) is greater than 600:1 and the ratio offree amino acid to anti-α4β7 antibody (mole:mole) is greater than 250:1.

In another aspect, the invention relates to a stable liquid formulationcomprising in aqueous solution with a non-reducing sugar, an anti-α4β7antibody and at least one free amino acid, wherein the molar ratio ofnon-reducing sugar to anti-α4β7 antibody (mole:mole) is greater than600:1. In yet a further aspect, the invention concerns a liquidformulation comprising at least about 40 mg/ml to about 80 mg/mlanti-α4β7 antibody, at least about 50-175 mM of one or more amino acids,and at least about 6% to at least about 10% (w/v) sugar. The liquidformulation may also contain a buffering agent. In some embodiments theliquid formulation also comprises a metal chelator. In some embodiments,the liquid formulation also comprises an anti-oxidant.

In another aspect, the invention relates to a liquid formulationcomprising at least about 60 mg/ml anti-α4β7 antibody, at least about10% (w/v) non-reducing sugar, and at least about 125 mM of one or morefree amino acids.

In another aspect, the invention relates to a liquid formulationcomprising at least about 60 mg/ml anti-α4β7 antibody, at least about10% (w/v) non-reducing sugar, and at least about 175 mM of one or morefree amino acids

In still yet a further aspect, the invention also relates to a dry,e.g., lyophilized formulation comprising a mixture of a non-reducingsugar, an anti-α4β7 antibody, histidine, arginine, and polysorbate 80,wherein the formulation is in solid form, and the molar ratio ofnon-reducing sugar to anti-α4β7 antibody (mole:mole) is greater than600:1.

In still yet a further aspect, the invention relates to a lyophilizedformulation comprising a mixture of a non-reducing sugar, an anti-α4β7antibody, histidine, arginine, and polysorbate 80. In this aspect, themolar ratio of non-reducing sugar to anti-α4β7 antibody (mole:mole) isgreater than 600:1. Furthermore, the molar ratio of arginine toanti-α4β7 antibody (mole:mole) in the formulation is greater than 250:1.

In another aspect, the invention relates to a method of making aformulation described herein, comprising maintaining the producttemperature below the collapse temperature during primary drying. Themethod can also contain an annealing step.

In one aspect, the invention relates to a method for treating a humanpatient suffering from inflammatory bowel disease, wherein the methodcomprises the step of administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to the following dosing regimen: (a) an initialdose of 300 mg of the humanized immunoglobulin or antigen-bindingfragment thereof as an intravenous infusion; (b) followed by a secondsubsequent dose of 300 mg of the humanized immunoglobulin orantigen-binding fragment thereof as an intravenous infusion at about twoweeks after the initial dose; (c) followed by a third subsequent dose of300 mg of the humanized immunoglobulin or antigen-binding fragmentthereof as an intravenous infusion at about six weeks after the initialdose; (d) followed by a fourth and subsequent doses of 300 mg of thehumanized immunoglobulin or antigen-binding fragment thereof as anintravenous infusion every four weeks or every eight weeks after thethird subsequent dose of the humanized antibody as needed: wherein thedosing regimen induces a clinical response and/or clinical remission inthe inflammatory bowel disease of the patient; and further wherein thehumanized immunoglobulin or antigen-binding fragment has bindingspecificity for the α4β7 complex, wherein the antigen-binding regioncomprises three complementarity determining regions (CDR1, CDR2, andCDR3) of a light chain variable region and three complementaritydetermining regions (CDR1, CDR2, and CDR3) of a heavy chain variableregion of the amino acid sequence set forth below: light chain: CDR1 SEQID NO: 11, CDR2 SEQ ID NO: 12, and CDR3 SEQ ID NO: 13; heavy chain: CDR1SEQ ID NO: 8, CDR2 SEQ ID NO: 9, and CDR3 SEQ ID NO: 10.

In another aspect, the invention relates to a dosing regimen for thetherapeutic treatment of inflammatory bowel disease, wherein the dosingregimen comprises the step of: administering to a patient suffering frominflammatory bowel disease, a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for humanα4β7 integrin, wherein the humanized immunoglobulin or antigen-bindingfragment comprises an antigen-binding region of nonhuman origin and atleast a portion of an antibody of human origin, wherein the humanizedimmunoglobulin or antigen-binding fragment thereof is administered tothe patient according to the following dosing regimen: (a) an initialdose of 300 mg of the humanized immunoglobulin or antigen-bindingfragment thereof as an intravenous infusion; (b) followed by a secondsubsequent dose of 300 mg of the humanized immunoglobulin orantigen-binding fragment thereof as an intravenous infusion at about twoweeks after the initial dose; (c) followed by a third subsequent dose of300 mg of the humanized immunoglobulin or antigen-binding fragmentthereof as an intravenous infusion at about six weeks after the initialdose; (d) followed by a fourth and subsequent doses of 300 mg of thehumanized immunoglobulin or antigen-binding fragment thereof as anintravenous infusion every four weeks or every eight weeks after thethird subsequent dose of the humanized antibody as needed; wherein thedosing regimen induces a clinical response and/or clinical remission inthe inflammatory bowel disease of the patient; and further wherein thehumanized immunoglobulin or antigen-binding fragment has bindingspecificity for the α4β7 complex, wherein the antigen-binding regioncomprises three complementarity determining regions (CDR1, CDR2, andCDR3) of a light chain variable region and three complementaritydetermining regions (CDR1, CDR2, and CDR3) of a heavy chain variableregion of the amino acid sequence set forth below: light chain: CDR1 SEQID NO: 11, CDR2 SEQ ID NO: 12, and CDR3 SEQ ID NO: 13; heavy chain: CDR1SEQ ID NO: 8, CDR2 SEQ ID NO: 9, and CDR3 SEQ ID NO:10.

In some aspects the method of treatment with the anti-α4β7 antibodyformulation, the dose, or the dose regimen can minimize immunogenicityof the anti-α4β7 antibody.

The patient may have had a lack of an adequate response with, lossresponse to, or was intolerant to treatment with at least one of animmunomodulator, a tumor necrosis factor-alpha (TNF-α) antagonist orcombinations thereof.

The inflammatory bowel disease can be Crohn's disease or ulcerativecolitis. The inflammatory bowel disease can be moderate to severelyactive ulcerative colitis.

The dosing regimen can result in mucosal healing in patients sufferingfrom moderate to severely active ulcerative colitis.

The patient may have previously received treatment with at least onecorticosteroid for the inflammatory bowel disease. The dosing regimencan result in a reduction, elimination or reduction and elimination ofcorticosteroid use by the patient.

In some aspects, the humanized immunoglobulin or antigen-bindingfragment thereof is administered in a final dosage form at aconcentration of between about 1.0 mg/ml to about 1.4 mg/ml. Thehumanized immunoglobulin or antigen-binding fragment thereof can beadministered in a final dosage form of about 1.2 mg/ml. The humanizedimmunoglobulin or antigen-binding fragment can be administered to thepatient in about 30 minutes.

The humanized immunoglobulin or antigen-binding fragment thereof can bereconstituted from a lyophilized formulation.

The humanized immunoglobulin or antigen-binding fragment thereof can bereconstituted to comprise a stable liquid formulation.

In some aspects, the dosing regimen does not alter the ratio of CD4 toCD8 in cerebrospinal fluid of patients receiving said treatment.

The patient can be a person 65 years of age or older and does notrequire any adjustment of the dosing regimen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a nucleotide sequence (SEQ ID NO:1)encoding the heavy chain of a humanized anti-α4β7 immunoglobulin, andthe deduced amino acid sequence of the heavy chain (SEQ ID NO:2). Thenucleotide sequence contains cloning sites (lower case), Kozak sequence(upper case, nucleotides 18-23 of SEQ ID NO:1) and leader sequence(lower case, nucleotides 24-86 of SEQ ID NO:1) at the 5′ end of theheavy chain. The open reading frame of the nucleotide sequence isnucleotides 24-1433 of SEQ ID NO:1.

FIG. 2 is an illustration of a nucleotide sequence (SEQ ID NO:3)encoding the light chain of a humanized immunoglobulin referred toherein as vedolizumab, and the deduced amino acid sequence (SEQ ID NO:4) of the light chain. The nucleotide sequence contains cloning sites(lower case), Kozak sequence (upper case, nucleotides 18-23 of SEQ IDNO:3) and leader sequence (lower case, nucleotides 24-80 of SEQ ID NO:3)at the 5′ end of the heavy chain. The open reading frame of thenucleotide sequence is nucleotides 24-737 of SEQ ID NO:3.

FIG. 3 is an alignment of the amino acid sequences of (A) the maturehumanized light chain (amino acids 20-238 of SEQ ID NO:4) of thehumanized immunoglobulin referred to herein as vedolizumab and (B) themature humanized light chain of the humanized immunoglobulin referred toherein as LDP-02 (SEQ ID NO:5). (Regarding LDP-02, see, WO 98/06248 andFeagan et al., N. Eng. J. Med. 352:2499-2507 (2005). Feagan et al.describe a clinical study of LDP-02, but in the article they refer toLDP-02 as MLN02.) The alignment illustrates that the amino acidsequences of the light chains of vedolizumab and LDP-02 differ atpositions 114 and 115 of the mature light chains.

FIG. 4 is an alignment of amino acid sequences of (A) a generic humankappa light chain constant region (SEQ ID NO:6) and (B) a generic murinekappa light chain constant region (SEQ ID NO:7). The amino acid residuesThr and Val (which are present at positions 114 and 115 of the maturevedolizumab light chain (amino acids 133 and 134 of SEQ ID NO:4)) arepresent in the constant region of the human kappa light chain, whereasthe amino acid residues Ala and Asp (which are present at positions 114and 115 of the mature LDP-02 light chain (SEQ ID NO:5)) are present inthe constant region of the mouse kappa light chain.

FIG. 5 is a map of vector pLKTOK38D (also referred to aspTOK38MLN02-TV), which encodes the humanized heavy chain and thehumanized light chain of MLN02, and is suitable for producingvedolizumab in CHO cells. (See, U.S. Patent Application Publication No.2004/0033561 A1 which discloses pLKTOK38. pLKTOK38D is a variant ofpLKTOK38 in which the restriction sites indicated on the map flank thesequence encoding the light chain variable region.)

FIG. 6A shows the predicted models for change in percent monomer, changein percent aggregate, and change in percent major isoform of theanti-α4β7 lyophilized formulation. The models are based on statisticalanalysis of the data presented in Example 1. The center line shows theresults for the predictive models and the outer lines show the 95%confidence limit for the predictive models. FIG. 6B shows alternativemodels based on the statistical analysis of 40° C. data from Tables 1-3when the input factors are pH, sugar:protein molar ratio, andarginine:protein molar ratio. The center line shows the results for thepredictive models and the outer lines show the 95% confidence limit forthe predictive models.

FIG. 7 shows the amino acid sequences of (A) the mature human GM607′CLantibody kappa light chain variable region and (B) the human 21/28′CLheavy chain variable region.

FIG. 8 is a graph showing that solids and load affect drying time (thenumbers in the lines represent the number of minutes of drying time).

FIG. 9 is a graph showing vedolizumab did not did not delay onset ofclinical symptoms of experimental autoimmune encephalomyelitis (EAE) ascompared to placebo control. Natalizumab significantly (p<0.05) delayedonset of clinical symptoms of EAE as compared to placebo control.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to formulations comprising anti-α4β7 antibodies.The formulations may be mixtures comprising non-reducing sugar,anti-α4β7 antibody and one or more free amino acids, and the molar ratioof the non-reducing sugar to anti-α4β7 antibody is greater than 600 molenon-reducing sugar:1 mole anti-α4β7 antibody. The formulations may be ina solid or liquid form.

Definitions

The term “pharmaceutical formulation” refers to a preparation thatcontains an anti-α4β7 antibody in such form as to permit the biologicalactivity of the antibody to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “stable” formulation is one in which the antibody thereinsubstantially retains its physical stability and/or chemical stabilityand/or its biological activity upon storage. In one aspect, theformulation substantially retains its physical and chemical stability,as well as its biological activity upon storage. The storage period isgenerally selected based on the intended shelf-life of the formulation.Various analytical techniques for measuring protein stability areavailable in the art and are reviewed in Peptide and Protein DrugDelivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), forexample.

A “deamidated” monoclonal antibody is one in which one or moreasparagine or glutamine residue thereof has been derivatized, e.g. to anaspartic acid or an iso-aspartic acid.

An antibody which is “susceptible to deamidation” is one comprising oneor more residue which has been found to be prone to deamidate.

An antibody which is “susceptible to oxidation” is an antibodycomprising one or more residue which has been found to be prone tooxidation.

An antibody which is “susceptible to aggregation” is one which has beenfound to aggregate with other antibody molecule(s), especially uponfreezing, heating, drying, reconstituting and/or agitation.

An antibody which is “susceptible to fragmentation” is one which hasbeen found to be cleaved into two or more fragments, for example at ahinge region thereof.

By “reducing deamidation, oxidation, aggregation, or fragmentation” isintended to mean preventing or decreasing (e.g., to 80%, 60%, 50%, 40%,30%, 20% or 10% of) the amount of deamidation, aggregation, orfragmentation relative to the monoclonal antibody formulated at adifferent pH or in a different buffer.

An “aggregate”, “SEC aggregate”, or “soluble aggregate” is more than oneand less than or equal to ten antibody proteins and/or fragmentsassociated together through either covalent, ionic, or hydrophobicinteractions to form a larger protein body.

An “insoluble aggregate” or “particle” is greater than ten antibodyproteins and/or fragments associated together through either covalent,ionic, or hydrophobic interactions to form a larger protein body.

As used herein, “biological activity” of a monoclonal antibody refers tothe ability of the antibody to bind to antigen and result in ameasurable biological response which can be measured in vitro or invivo. Such activity may be antagonistic or agonistic.

The cell surface molecule, “α4β7 integrin,” or “α4β7,” is a heterodimerof an α₄ chain (CD49D, ITGA4) and a β₇ chain (ITGB7). Each chain canform a heterodimer with an alternative integrin chain, to form α₄β₁ orα_(E)β₇. Human α₄ and β₇ genes (GenBank (National Center forBiotechnology Information, Bethesda, Md.) RefSeq Accession numbers NM000885 and NM 000889, respectively) are expressed by B and Tlymphocytes, particularly memory CD4+ lymphocytes. Typical of manyintegrins, α4β7 can exist in either a resting or activated state.Ligands for α4β7 include vascular cell adhesion molecule (VCAM),fibronectin and mucosal addressin (MAdCAM (e.g., MAdCAM-1)).

As used herein, a human immunoglobulin or antigen-binding fragmentthereof that has “binding specificity for the α4β7 complex” binds toα4β7, but not to α4β1 or αEB7.

As used herein, an “isotonic” formulation has substantially the sameosmotic pressure as human blood. Isotonic formulations will generallyhave an osmotic pressure from about 250 to 350 mOsm. Isotonicity can bemeasured using a vapor pressure or ice-freezing type osmometer, forexample.

As used herein, “buffering agent” refers to a buffer that resistschanges in pH by the action of its acid-base conjugate components. Thebuffering agent may be present in a liquid or solid formulation of theinvention. The buffering agent adjusts the pH of the formulation toabout 5.0 to about 7.5, to about 5.5 to about 7.5, to about 6.0 to about6.5, or to a pH of about 6.3. In one aspect, examples of bufferingagents that will control the pH in the 5.0 to 7.5 range include acetate,succinate, gluconate, histidine, citrate, phosphate, maleate,cacodylate, 2-[N-morpholino]ethanesulfonic acid (MES),bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris),N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine and otherorganic acid buffers. In another aspect, the buffering agent herein ishistidine or citrate.

A “histidine buffer” is a buffer comprising histidine ions. Examples ofhistidine buffers include histidine chloride, histidine acetate,histidine phosphate, histidine sulfate solutions. The histidine bufferor histidine-HCl buffer has a pH between about pH 5.5 to 6.5, about pH6.1 to 6.5, or about pH 6.3.

A “saccharide” herein is a compound that has a general formula(CH₂O)_(n) and derivatives thereof, including monosaccharides,disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducingsugars, nonreducing sugars, and the like. In one aspect, examples ofsaccharides herein include glucose, sucrose, trehalose, lactose,fructose, maltose, dextran, erythritol, glycerol, arabitol, sylitol,sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose,stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol,iso-maltulose, and the like. A saccharide can be a lyoprotectant. Inanother aspect, the saccharide herein is a nonreducing disaccharide,such as sucrose.

A “surfactant” herein refers to an agent that lowers surface tension ofa liquid. The surfactant can be a nonionic surfactant. In one aspect,examples of surfactants herein include polysorbate (polyoxyethylenesorbitan monolaurate, for example, polysorbate 20 and, polysorbate 80);TRITON (t-Octylphenoxypolyethoxyethanol, nonionic detergent, UnionCarbide subsidiary of Dow Chemical Co., Midland Mich.); sodium dodecylsulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; sorbitan monopalmitate; and the MONAQUAT series(Mona Industries, Inc., Paterson, N.J.); polyethyl glycol (PEG),polypropylene glycol (PPG), and copolymers of poloxyethylene andpoloxypropylene glycol (e.g. Pluronics/Poloxamer, PF68 etc); etc. Inanother aspect, the surfactant is polysorbate 80.

The term “antibody” herein is used in the broadest sense andspecifically covers full length monoclonal antibodies, immunoglobulins,polyclonal antibodies, multispecific antibodies (e.g. bispecificantibodies) formed from at least two full length antibodies, e.g., eachto a different antigen or epitope, and individual antigen bindingfragments, including dAbs, scFv, Fab, F(ab)′₂, Fab′, including human,humanized and antibodies from non-human species and recombinant antigenbinding forms such as monobodies and diabodies.

Molar amounts and ratios of anti-α4β7 antibody to other excipientsdescribed herein are calculated on the assumption of an approximatemolecular weight of about 150,000 daltons for the antibody. The actualantibody molecular weight may differ from 150,000 daltons, depending onamino acid composition or post-translational modification, e.g., asdependent on the cell line used to express the antibody. Actual antibodymolecular weight can be +/−5% of 150,000 daltons.

The term “human antibody” includes an antibody that possesses a sequencethat is derived from a human germ-line immunoglobulin sequence, such asan antibody derived from transgenic mice having human immunoglobulingenes (e.g., XENOMOUSE genetically engineered mice (Abgenix, Fremont,Calif.), HUMAB-MOUSE®, KIRIN TC MOUSE™ transchromosome mice, KMMOUSE®(MEDAREX, Princeton, N.J.)), human phage display libraries, humanmyeloma cells, or human B cells.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen binding sequences derived from a non-humanprimate (e.g. Old World Monkey, Ape etc) and human constant regionsequences.

“Antigen binding fragments” of the humanized immunoglobulin prepared inthe formulation of the invention comprise at least the variable regionsof the heavy and/or light chains of an anti-α4β7 antibody. For example,an antigen binding fragment of vedolizumab comprises amino acid residues20-131 of the humanized light chain sequence of SEQ ID NO:4. Examples ofsuch antigen binding fragments include Fab fragments, Fab′ fragments,scFv and F(ab′)₂ fragments of a humanized immunoglobulin known in theart. Antigen binding fragments of the humanized immunoglobulin of theinvention can be produced by enzymatic cleavage or by recombinanttechniques. For instance, papain or pepsin cleavage can be used togenerate Fab or F(ab′)₂ fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons have been introduced upstream of the naturalstop site. For example, a recombinant construct encoding the heavy chainof an F(ab′)₂ fragment can be designed to include DNA sequences encodingthe CH₁ domain and hinge region of the heavy chain. In one aspect,antigen binding fragments inhibit binding of α4β7 integrin to one ormore of its ligands (e.g. the mucosal addressin MAdCAM (e.g., MAdCAM-1),fibronectin).

Papain digestion of antibodies produces two identical antigen bindingfragments, called “Fab” fragments, each with a single antigen bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding sites and is still capable of cross-linkingantigen.

“Fv” is an antibody fragment which consists of a dimer of one heavychain variable domain and one light chain variable domain innon-covalent association.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In one aspect, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with two antigenbinding sites, which fragments comprise a variable heavy domain (V_(H))connected to a variable light domain (V_(L)) in the same polypeptidechain (V_(H)-V_(L)). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “full length antibody” is one which comprises an antigen bindingvariable region as well as a light chain constant domain (C_(L)) andheavy chain constant domains, C_(H1), C_(H2) and C_(H3). The constantdomains may be native sequence constant domains (e.g. human nativesequence constant domains) or amino acid sequence variants thereof. Inone aspect, the full length antibody has one or more effector functions.

An “amino acid sequence variant” antibody herein is an antibody with anamino acid sequence which differs from a main species antibody.Ordinarily, amino acid sequence variants will possess at least about70%, at least about 80%, at least about 85%, at least about 90%, or atleast about 95% homology with the main species antibody. The amino acidsequence variants possess substitutions, deletions, and/or additions atcertain positions within or adjacent to the amino acid sequence of themain species antibody, but retain antigen binding activity. Variationsin sequence of the constant regions of the antibody will have lesseffect on the antigen binding activity than variations in the variableregions. In the variable regions, amino acid sequence variants will beat least about 90% homologous, at least about 95% homologous, at leastabout 97% homologous, at least about 98% homologous, or at least about99% homologous with the main species antibody.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart.

A “therapeutic monoclonal antibody” is an antibody used for therapy of ahuman subject. Therapeutic monoclonal antibodies disclosed hereininclude anti-α4β7 antibodies.

A “glycosylation variant” antibody herein is an antibody with one ormore carbohydrate moeities attached thereto which differ from one ormore carbohydrate moieties attached to a main species antibody. Examplesof glycosylation variants herein include antibody with a G1 or G2oligosaccharide structure, instead of a G0 oligosaccharide structure,attached to an Fc region thereof, antibody with one or two carbohydratemoieties attached to one or two light chains thereof, antibody with nocarbohydrate attached to one or two heavy chains of the antibody, etc,and combinations of glycosylation alterations.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), and the like.

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to different“classes”. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one aspect, the FcR is anative sequence human FcR. In another aspect, the FcR is one which bindsan IgG antibody (a gamma receptor) and includes receptors of the FcγRI,FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (See review in M. Daeron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:33-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined. The hypervariableregion or the CDRs thereof can be transferred from one antibody chain toanother or to another protein to confer antigen binding specificity tothe resulting (composite) antibody or binding protein.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable regions thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). In one aspect, affinitymatured antibodies will have nanomolar or even picomolar affinities forthe target antigen. Affinity matured antibodies are produced byprocedures known in the art. Marks et al. Bio/Technology 10:779-783(1992) describes affinity maturation by VH and VL domain shuffling.Random mutagenesis of CDR and/or framework residues is described by:Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier etal. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins etal., J. Mol. Biol. 226:889-896 (1992).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment. In certainembodiments, the antibody will be purified (1) to greater than 95% byweight of protein as determined by the Lowry method, and alternatively,more than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, isolated antibody will be preparedby at least one purification step.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disease as well as those in which the disease or its recurrenceis to be prevented. Hence, the patient to be treated herein may havebeen diagnosed as having the disease or may be predisposed orsusceptible to the disease. The terms “patient” and “subject” are usedinterchangeably herein.

The antibody which is formulated is substantially pure and desirablysubstantially homogeneous (i.e. free from contaminating proteins etc).“Substantially pure” antibody means a composition comprising at leastabout 90% antibody by weight, based on total weight of the protein inthe composition, at least about 95% or 97% by weight. “Substantiallyhomogeneous” antibody means a composition comprising protein wherein atleast about 99% by weight of protein is specific antibody, e.g.,anti-α4β7 antibody, based on total weight of the protein.

“Clinical remission” as used herein with reference to ulcerative colitissubjects refers to a complete Mayo score of 2 or less points and noindividual subscore greater than 1 point. Crohn's disease “clinicalremission” refers to a CDAI score of 150 points or less.

A “clinical response” as used herein with reference to ulcerativecolitis subjects refers to a reduction in complete Mayo score of 3 orgreater points and 30% from baseline, (or a partial Mayo score of 2 orgreater points and 25% or greater from baseline, if the complete Mayoscore was not performed at the visit) with an accompanying decrease inrectal bleeding subscore of 1 or greater points or absolute rectalbleeding score of 1 or less point. A “clinical response” as used hereinwith reference to Crohn's disease subjects refers to a 70 point orgreater decrease in CDAI score from baseline (week 0).

“Mucosal healing” as used herein with reference to ulcerative colitissubjects refers to an endoscopic subscore of 1 point or less.

As used herein, “treatment failure” refers to disease worsening, a needfor rescue medications or surgical intervention for treatment ofulcerative colitis or Crohn's disease. A rescue medication is any newmedication or any increase in dose of a baseline medication required totreat new or unresolved ulcerative colitis or Crohn's disease symptoms(other than antidiarrheals for control of chronic diarrhea).

Formulations

As described herein, it has been discovered that anti-α4β7 antibodiesare highly stable when in a dry, e.g., lyophilized formulation withexcess (on a mole basis) non-reducing sugar. In particular, lyophilizedformulations in which the ratio of non-reducing sugar to anti-α4β7antibody (mole:mole) is greater than 600:1 are shown herein to be stablefor at least 2 years.

The present invention provides, in a first aspect, a stable anti-α4β7antibody formulation. In one aspect, the formulation comprises a buffer,at least one stabilizer and an anti-α4β7 antibody. In one aspect, a dryformulation comprises one or more non-reducing sugars and an anti-α4β7antibody, wherein the ratio of non-reducing sugar to anti-α4β7 antibody(mole:mole) is greater than 600:1. The formulation also comprises one ormore free amino acids. One or more of the amino acids also can act as abuffer. In one aspect, one or more of the amino acids can act as astabilizer. The formulation may optionally further comprise at least onesurfactant. In one embodiment, the formulation is dry, e.g.,lyophilized. The antibody in the formulation may be a full lengthantibody or an antigen binding fragment thereof, such as a Fab, Fv,scFv, Fab′ or F(ab′)₂ fragment.

The formulation can contain any desired non-reducing sugars. In oneaspect, non-reducing sugars that can be included in the formulationinclude, for example, mannitol, sorbital, sucrose, trehalose, raffinose,stachyose, melezitose, dextran, maltitol, lactitol, isomaltulose,palatinit and combinations thereof. In another aspect, non-reducingsugars are sucrose, trehalose, mannitol, and sorbitol. The absoluteamount of non-reducing sugar in the formulation is not critical, but theratio of non-reducing sugar to anti-α4β7 antibody (mole:mole) is greaterthan 400:1 In another aspect, the ratio of non-reducing sugar toanti-α4β7 antibody (mole:mole) is at least about 600:1; at least about625:1; at least about 650:1; at least about 675:1, at least about 700:1;at least about 750:1, at least about 800:1, at least about 1000:1, atleast about 1200:1, at least about 1400:1, at least about 1500:1, atleast about 1600:1, at least about 1700:1, at least about 1800:1, atleast about 1900:1, or at least about 2000:1. Generally, it is desirablethat the non-reducing sugar is present in an amount which reducessoluble aggregate formation in a liquid formulation, such as aggregateformation which occurs upon freezing and thawing and/or drying andreconstituting. A ratio of non-reducing sugar to anti-α4β7 antibody(mole:mole) higher than about 730:1 may give slightly reduced solubleaggregate formation in the lyophilized state. The sugar:protein weightratio can be greater than 1.5:1 (w/w). In another aspect, thenon-reducing sugar concentrations for liquid (e.g., pre-drying orpost-reconstitution) formulations are in the range from about 10 mM toabout 1 M, for example, from about 60 mM to about 600 mM, about 100 mMto about 450 mM, about 200 mM to about 350 mM, about 250 mM to about 325mM, and about 275 mM to about 300 mM. In another aspect, the amounts ofnon-reducing sugar in a dry, (e.g., lyophilized) formulation are in therange from about 40% to about 70% (w/w of dry formulation). In anotheraspect, the amounts of non-reducing sugar in a dry (e.g., lyophilized)formulation are in the range from about 40% to about 60%, from about 45%to about 55% or about 51% (w/w). In other aspects, the amount ofnon-reducing sugar in a dry, (e.g., lyophilized) formulation is greaterthan about 51% (w/w of dry formulation) when the protein amount is about31% (w/w of dry formulation) or greater than about a 1.6:1 mass ratio ofnon-reducing sugar to protein in the dry formulation. In yet stillanother aspect, sucrose is the non-reducing sugar for use in theformulation.

The formulation can contain any desired free amino acid, which can be inthe L-form, the D-form or any desired mixture of these forms. In oneaspect, free amino acids that can be included in the formulationinclude, for example, histidine, alanine, arginine, glycine, glutamicacid, serine, lysine, tryptophan, valine, cysteine and combinationsthereof. Some amino acids can stabilize the proteins against degradationduring manufacturing, drying, lyophilization and/or storage, e.g.,through hydrogen bonds, salt bridges antioxidant properties orhydrophobic interactions or by exclusion from the protein surface. Aminoacids can act as tonicity modifiers or can act to decrease viscosity ofthe formulation. In another aspect, free amino acids, such as histidineand arginine, can act as cryoprotectants and lyoprotectants, and do notcrystallize when lyophilized as components of the formulation. Freeamino acids, such as glutamic acid and histidine, alone or incombination, can act as buffering agents in aqueous solution in the pHrange of 5 to 7.5. In still yet another aspect, the formulation containshistidine, or histidine and arginine. In still yet a further aspect, thefree amino acid concentrations for liquid formulations are in the rangefrom about 10 mM to about 0.5 M, for example, from about 15 mM to about300 mM, about 20 mM to about 200 mM, or about 25 mM to about 150 mM,about 50 mM or about 125 mM. In still yet a further aspect, the amountsof histidine in a dry, (e.g., lyophilized) formulation are in the rangefrom about 1% to about 10% (w/w of dry formulation), or from about 3% toabout 6% (w/w). In some embodiments, the amount of histidine in a dry,(e.g., lyophilized) formulation is greater than about 4% (w/w of the dryformulation) when the protein amount is about 31% (w/w of the dryformulation) or greater than about a 0.15:1 mass ratio of histidine toprotein in the dry formulation. In still yet another aspect, the amountsof arginine in a dry, (e.g., lyophilized) formulation are in the rangefrom about 4% to about 20% (w/w of dry formulation), or from about 10%to about 15% (w/w). In some embodiments, the amount of arginine in adry, (e.g., lyophilized) formulation is greater than about 13% (w/w ofthe dry formulation) when the protein amount is about 31% (w/w of thedry formulation) or greater than about a 0.4:1 mass ratio of arginine toprotein in the dry formulation. In embodiments of combinations of aminoacids, such as histidine and arginine, the molar ratio of total aminoacid to antibody ratio can be at least 200:1, about 200:1 to about500:1, or at least 400:1.

The formulation can optionally further contain at least one surfactant.In one aspect, surfactants that can be included in the formulationinclude, for example, polysorbate 20, polysorbate 80, a poloxamer(Pluronic®) and combinations thereof. When present, the surfactant isgenerally included in an amount which reduces formation of insolubleaggregates of antibody, e.g., during bottling, freezing, drying,lyophilization and/or reconstitution. The surfactant concentration,e.g., in a pre-dry, (e.g., lyophilized) or post-reconstitutionformulation, is generally from about 0.0001% to about 1.0%, from about0.01% to about 0.1%, for example about 0.02%, 0.03%, 0.04%, 0.05%,0.06%, 0.07%, 0.08,% or 0.09% (w/v), 0.05% to 0.07% or 0.06% (w/v). Thesurfactant amount, e.g., in a dry, (e.g., lyophilized) formulation, isgenerally from about 0.01% to about 3.0% (w/w), from about 0.10% toabout 1.0%, for example about 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%,or 0.50% (w/w). In another aspect, the surfactant:antibody molar ratiois about 1:1. The anti-α4β7 antibody can be present in any desiredamount in the formulation, provided that the ratio of non-reducing sugarto anti-α4β7 antibody (mole:mole) is greater than about 600:1. However,the formulation can contain a high concentration of anti-α4β7 antibody.For example, liquid formulations can comprise at least about 10 mg/ml,at least about 20 mg/ml, at least about 30 mg/ml, at least about 40mg/ml, at least about 50 mg/ml, at least about 60 ml/ml, at least about70 mg/ml, at least about 80 mg/ml, at least about 90 mg/ml, at leastabout 100 mg/ml, from about 40 mg/ml to about 80 mg/ml anti-α4β7antibody, about 60 mg/ml anti-α4β7 antibody. Dry formulations (e.g.,lyophilized) can contain at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,or about 31% or about 32% anti-α4β7 antibody by weight.

If desired, the formulation can further comprise a metal chelator and/oran anti-oxidant, as well as other pharmaceutically acceptableexcipients. Suitable metal chelators include, for example, methylamine,ethylenediamine, desferoxamine, trientine, histidine, malate,phosphonate compounds, e.g., etidronic acid, ethylenediaminetetraaceticacid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and the like.Suitable anti-oxidants include, for example, citric acid, uric acid,ascorbic acid, lipoic acid, glutathione, tocopherol, carotene, lycopene,cysteine and the like.

The formulation can be a liquid or a solid. Liquid formulations can beaqueous solutions or suspensions, prepared in a suitable aqueoussolvent, such as water or an aqueous/organic mixture, such as wateralcohol mixtures. Liquid formulations can have a pH between about 5.5and about 7.5, between about 6.0 and about 7.0, or between about 6.0 andabout 6.5, such as about 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5. Liquidformulations can be refrigerated (e.g., 2-8° C.) or frozen (e.g., at−20° C. or −80° C.) for storage. Solid formulations can be prepared inany suitable way and can be in the form of a cake or powder, forexample. The solid formulation is prepared by drying a liquidformulation as described herein, for example by lyophilization, spraydrying, air drying in a film (e.g., for transdermal delivery), mixinginto a lipid emulsion and drying as spheres for oral delivery or filmfor transdermal delivery. When the formulation is a solid formulation,the formulation can have a moisture content of no more than about 5%, nomore than about 4.5%, no more than about 4%, no more than about 3.5%, nomore than about 3%, no more than about 2.5%, no more than about 2%, nomore than about 1.5%, no more than about 1%, or is substantiallyanhydrous. Solid formulations can be dissolved, i.e. reconstituted, in asuitable medium or solvent to become liquid suitable for administration.Suitable solvents for reconstituting the solid formulation includewater, isotonic saline, buffer, e.g., phosphate-buffered saline,Ringer's (lactated or dextrose) solution, minimal essential medium,alcohol/aqueous solutions, dextrose solution, etc. The amount of solventcan result in a therapeutic protein concentration higher, the same, orlower than the concentration prior to drying. In one aspect, thereconstituted anti-α4β7 antibody concentration is the same concentrationas in the pre-drying liquid formulation.

The formulation may be sterile, and this can be achieved according tothe procedures known to the skilled person for generating sterilepharmaceutical formulations suitable for administration to humansubjects, prior to, or following, preparation of the formulation. Theformulation can be sterilized as a liquid, e.g., before drying and/orafter reconstitution by filtration through small pores, through asepticprocessing or by exposure to ultraviolet radiation. Filter pore sizescan be 0.1 μm or 0.2 μm to filter microorganisms or 10 to 20 nm tofilter virus particles. Alternatively, or additionally, the driedformulation can be sterilized, e.g., by exposure to gamma radiation. Inone aspect, the anti-α4β7 antibody liquid formulation is sterilized byfiltration before drying.

In one aspect, the formulation is stable upon storage. In anotheraspect, the formulation is stable upon storage in the dry state.Stability can be tested by evaluating physical stability, chemicalstability, and/or biological activity of the antibody in the formulationaround the time of formulation as well as following storage at the notedtemperatures. Physical and/or chemical stability of a liquid formulationor a reconstituted dry powder can be evaluated qualitatively and/orquantitatively in a variety of different ways (see, e.g., AnalyticalTechniques for Biopharmaceutical Development, Rodriguez-Diaz et al. eds.Informa Healthcare (2005)), including evaluation of aggregate formation(for example using size exclusion (or gel filtration) chromatography(SEC), matrix-assisted laser desorption-ionization time-of-flight massspectrometry (MALDI-TOF MS), analytical ultracentrifugation, lightscattering (photon correlation spectroscopy, dynamic light scattering(DLS), multi-angle laser light scattering (MALLS)), flow-basedmicroscopic imaging, electronic impedance (coulter) counting, lightobscuration or other liquid particle counting system, by measuringturbidity, by density gradient centrifugation and/or by visualinspection); by assessing charge heterogeneity using cation exchangechromatography (see also Vlasak and Ionescu, Curr. Pharm. Biotechnol.9:468-481 (2008) and Harris et al. J. Chromatogr. B Biomed. Sci. Appl.752:233-245 (2001)), isoelectric focusing (IEF), e.g. capillarytechnique (cIEF), or capillary zone electrophoresis; amino-terminal orcarboxy terminal sequence analysis; mass spectrometric analysis;SDS-PAGE or SEC analysis to compare fragmented, intact and multimeric(i.e., dimeric, trimeric, etc.) antibody; peptide map (for exampletryptic or LYS- and the like); evaluating biological activity or antigenbinding function of the antibody; and the like. Biological activity orantigen binding function, e.g., binding of the anti-α4β7 antibody toMAdCAM (e.g., MAdCAM-1) or inhibition of the binding of a cellexpressing α4β7 integrin to MAdCAM (e.g., MAdCAM-1), e.g., immobilizedMAdCAM (e.g., MAdCAM-1), can be evaluated using various techniquesavailable to the skilled practitioner (see e.g., Soler et al., J.Pharmacol. Exper. Ther. 330:864-875 (2009)).

Stability of a solid-state formulation can also be evaluatedqualitatively and/or quantitatively in a variety of different ways,including direct tests, such as identifying crystal structure by X-RayPowder Diffraction (XRPD); evaluating antibody structure in the solidstate using Fourier Transform Infrared Spectroscopy (FTIR); andmeasuring thermal transitions in the lyophilized solid (melting, glasstransition, etc.) using Differential Scanning calorimetry (DSC, e.g., toassess denaturation) and indirect tests such as measuring moisturecontent by Karl Fisher test, e.g., to extrapolate the likelihood ofchemical instability through hydrolysis. Measurement of the moisturecontent of a dry formulation can indicate how likely a formulation willundergo chemical or physical degradation, with higher moisture leadingto more degradation.

Stability can be measured at a selected temperature for a selected timeperiod. In one aspect, a dry, (e.g., lyophilized) formulation is stableat about 40° C., 75% RH for at least about 2-4 weeks, at least about 2months, at least about 3 months, at least about 6 months, at least about9 months, at least about 12 months, or at least about 18 months. Inanother aspect, the formulation (liquid or dry (e.g., lyophilized)) isstable at about 5° C. and/or 25° C. and 60% RH for at least about 3months, at least about 6 months, at least about 9 months, at least about12 months, at least about 18 months, at least about 24 months, at leastabout 30 months, at least about 36 months, or at least about 48 months.In another aspect, the formulation (liquid or dry (e.g., lyophilized))is stable at about −20° C. for at least about 3 months, at least about 6months, at least about 9 months, at least about 12 months, at leastabout 18 months, at least about 24 months, at least about 30 months, atleast about 36 months, at least about 42 months, or at least about 48months. Furthermore, the liquid formulation may, in some embodiments, bestable following freezing (to, e.g., −80° C.) and thawing, such as, forexample, following 1, 2 or 3 cycles of freezing and thawing.

Instability may involve any one or more of: aggregation (e.g.,non-covalent soluble aggregation (caused by hydrophobic or chargeinteractions), covalent soluble aggregation (e.g., disulfide bondrearrangement/scrambling), insoluble aggregation (cause by denaturing ofthe protein at the liquid/air and liquid/solid interfaces)), deamidation(e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization(e.g. Asp isomeriation), denaturation, clipping/hydrolysis/fragmentation(e.g. hinge region fragmentation), succinimide formation, N-terminalextension, C-terminal processing, glycosylation differences, and thelike.

A stable formulation can contribute to a low immunogenicity of ananti-α4β7 antibody. An immunogenic anti-α4β7 antibody can lead to ahuman-anti-human antibody (HAHA) response in human subjects or patients.Patients who develop a HAHA response to an anti-α4β7 antibody can haveadverse events (e.g., site infusion reaction) upon treatment or caneliminate anti-α4β7 antibody quickly, resulting in a lower dose thanplanned by treatment. A report (Feagen et al. (2005) N. Engl. J. Med.352:2499-2507) of early study of an anti-α4β7 antibody treatmentindicated that human antihuman antibodies developed by week 8 in 44% oftreated patients. The antibody in this study was stored as a liquid anddid not contain any polysorbate.

In some embodiments, the formulation can increase the proportion of HAHAnegative patients to at least 40%, at least 50%, at least 60%, at least70%, at least 80% or at least 90% of patients compared to the HAHAresults of a less stable formulation.

In some embodiments, an anti-α4β7 antibody formulation has 50% majorcharged isoform, ≧55% major charged isoform, or 65 to 70% major chargedisoform. In other aspects, a stable anti-α4β7 antibody formulation has≦45% acidic charged isoforms, ≦40% acidic charged isoforms, ≦30% acidiccharged isoforms or 22 to 28% acidic isoforms. In still other aspects, astable anti-α4β7 antibody formulation has ≦25% basic isoforms, ≦20%basic isoforms, ≦15% basic isoforms, about 5% basic isoforms or about10% basic isoforms. In one aspect, a stable anti-α4β7 antibodyformulation has 55% major isoform, ≦30% acidic isoforms and/or ≦20%basic isoforms, e.g., as determined by CEX. In another aspect, a stableanti-α4β7 antibody formulation has ≧50% major isoform, ≦45% acidicisoforms and/or <10% basic isoforms, e.g., as determined by cIEF.

In some aspects, an anti-α4β7 antibody dry, solid formulation has ≦10%moisture content, ≦5% moisture content or ≦2.5% moisture content. Thetime required for reconstitution is ≦60 minutes, ≦50 minutes or ≦40minutes or ≦30 minutes or ≦20 minutes.

Monomeric content and/or aggregate content (e.g., as dimers, trimers,tetramers, pentamers, oligomers and higher-order aggregates), i.e., inthe liquid formulation, or in a dry formulation after reconstitution,can be measured by SEC, MALDI-TOF MS, analytical ultracentrifugation,light scattering (DLS or MALLS), or nanoscale measurement, such asnanoparticle tracking analysis NTA, NanoSight Ltd, Wiltshire, UK).Resolution, characterization and quantification of aggregate can beachieved in a number of ways, including increasing the length of the SECcolumn separation, e.g., by a longer column or by serial attachment of asecond or more SEC column(s) in line with the initial analytical SECcolumn, supplementing SEC quantification of monomers with lightscattering, or by using NTA.

In one embodiment, an anti-α4β7 antibody formulation has 90% monomericantibody, ≧95% monomeric antibody, or 97 to 99% monomeric antibody. Inanother embodiment, the majority of the material in an anti-α4β7antibody formulation has an average radius of ≦20 nm, ≦15 nm, ≦10 nm, orabout 5 to about 7 nm. In one aspect, an anti-α4β7 antibody formulationhas 80% amount heavy plus light chain by protein analysis. In oneaspect, there is 90% heavy plus light chain. In another aspect, ananti-α4β7 antibody formulation has ≦10% aggregate, ≦5% aggregate, ≦2.5%aggregate ≦1.5% aggregate, ≦1.0% aggregate or ≦0.5% aggregate. Inanother aspect, a stable anti-α4β7 antibody formulation has ≧96% monomerand/or ≦2.5% aggregate. In yet another aspect, a stable anti-α4β7antibody formulation has about 99% monomer and/or about <1% aggregate.

Particle sizes, e.g., of aggregates or undissolved excipient, i.e., inreconstituted formulation can be measured by light obscuration (e.g.,liquid particle counting system (HIAC) by Hach Ultra Analytics (GrantsPass, Oreg.)), microscopy, coulter counter, or digital (e.g.,flow-based) microscopic imaging based system such as microfluidicsimaging (MFI) by Brightwell (Ottawa, Calif.) or FLOWCAM® Image particleanalyzer by Fluid Imaging Technologies (Yarmouth, Me.). In one aspect,particle size in an anti-α4β7 antibody preparation is about 30 μM, about25 about 10 about 5 μm, about 2 μm or 1 μm or less. The amount ofparticles should be minimized in antibody formulations. In one aspect,the anti-α4β7 antibody formulation has less than 6000 particles ≧10 μMand less than 600 particles ≧25 μm diameter in one dose (U.S.Pharmacopoeia Chp. 788, light obscuration counting method; half thoseamounts by microscopic quantification method). In yet another aspect, anamount of particles per milliliter, e.g., by MFI measurement, in a doseof an anti-α4β7 antibody formulation, e.g., reconstituted formulation isabout 500 to about 2000, or about 1000 to about 3000 of 2-10 μmparticles per ml, about 50 to about 350 of ≧10 μm particles per ml andabout 0 to about 50 of ≧25 μm particles per ml.

In one embodiment, an anti-α4β7 antibody formulation has a bindingaffinity of about 60% to about 140% of the reference standard anti-α4β7antibody. In one aspect, an anti-α4β7 antibody in a formulationdescribed herein binds to α4β7, e.g., on a cell (WO98/06248 or U.S. Pat.No. 7,147,851), at a value of about 80% to about 120% of the referencestandard. In another embodiment, an anti-α4β7 antibody formulation hasthe ability to inhibit at least 50% or at least 60% of the binding of acell expressing α4β7 integrin to MAdCAM, e.g., MAdCAM-1, a MAdCAM-Igchimera (see U.S. Patent Application Publication No. 20070122404, alsofor reference standard examples).

As noted above, freezing of the formulation is specifically contemplatedherein. Hence, the formulation can be tested for stability upon freezingand thawing. Accordingly, the antibody in a liquid formulation may bestable upon freezing and thawing the formulation, for example theantibody can be stable after one, two, three, four, five or morefreeze/thaw cycles.

In some embodiments, the formulation is a liquid formulation comprisingat least about 50 mg/ml to about 100 mg/ml anti-α4β7 antibody, abuffering agent (e.g., histidine), and at least about 9% (w/w)non-reducing sugar (e.g., sucrose, trehalose or mannitol). In oneembodiment, the formulation comprises at least about 50 to about 80mg/ml, about 60 mg/ml anti-α4β7 antibody, a buffering agent (e.g.,histidine), a free amino acid (e.g., arginine) and at least about 9% or10% (w/w) non-reducing sugar (e.g., sucrose, trehalose or mannitol).

In another embodiment, the formulation comprises at least about 60 mg/mlanti-α4β7 antibody, a buffering agent (e.g., histidine), a free aminoacid (e.g., arginine) and at least about 10% (w/w) non-reducing sugar(e.g., sucrose, trehalose or mannitol). In such embodiments, the bufferconcentration is about 15 to about 75 mM, about 25 to about 65 mM, orabout 50 mM. The free amino acid concentration is about 50 to about 250mM, about 75 to about 200 mM, about 100 to about 150 mM or about 125 mM.

In one embodiment, the formulation is a dry, solid formulation (e.g., alyophilized formulation), comprising a mixture of a non-reducing sugar,an anti-α4β7 antibody, histidine, arginine, and polysorbate 80, and themolar ratio of non-reducing sugar to anti-α4β7 antibody (mole:mole) isgreater than 600:1.

In another embodiment, the formulation is a dry, solid, amorphousformulation (e.g., a lyophilized formulation), comprising a mixture of anon-reducing sugar, an anti-α4β7 antibody, histidine, arginine, andpolysorbate 80, and the molar ratio of non-reducing sugar to anti-α4β7antibody (mole:mole) is greater than 600:1.

In one embodiment, the formulation is a lyophilized formulationcomprising a non-reducing sugar, an anti-α4β7 antibody, histidine,arginine and polysorbate 80, and the molar ratio of non-reducing sugarto anti-α4β7 antibody (mole:mole) in the formulation is greater than600:1.

In one embodiment, the formulation is a lyophilized formulationcomprising a non-reducing sugar, an anti-α4β7 antibody, histidine,arginine and polysorbate 80, wherein the molar ratio of non-reducingsugar to anti-α4β7 antibody (mole:mole) in the formulation is greaterthan 600:1 and the molar ratio of arginine to anti-α4β7 antibody(mole:mole) in the formulation is greater than 250:1.

In one embodiment, the formulation is a liquid formulation and comprisesat least about 60 mg/ml anti-α4β7 antibody, at least about 10% (w/v)non-reducing sugar, and at least about 125 mM of one or more free aminoacids.

In one embodiment, the formulation is a liquid formulation and comprisesat least about 60 mg/ml anti-α4β7 antibody, at least about 10% (w/v)non-reducing sugar, and at least about 175 mM of one or more free aminoacids.

In one embodiment, the formulation is a liquid formulation and comprisesbetween about 60 mg/ml to about 80 mg/ml anti-α4β7 antibody, a bufferingagent and at least about 10% (w/w) sugar.

In one embodiment, the formulation is a liquid formulation and comprisesbetween about 60 mg/ml to about 80 mg/ml anti-α4β7 antibody, histidineand at least about 10% (w/w) sucrose.

In one embodiment, the formulation is lyophilized and stored as a singledose in one vial. The vial is desirably stored at about 2-8° C. until itis administered to a subject in need thereof. The vial may for examplebe a 20 or 50 cc vial (for example for a 60 mg/ml dose). The vial maycontain at least about 120 mg, at least about 180 mg, at least about 240mg, at least about 300 mg, at least about 360 mg, at least about 540 mg,or at least about 900 mg of anti-α4β7 antibody. In one aspect, the vialcontains about 300 mg of anti-α4β7 antibody.

One or more other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington: The Science andPractice of Pharmacy, 21st Edition, Hendrickson, R. Ed. (2005) may beincluded in the formulation provided that they do not adversely affectthe desired characteristics of the formulation. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed and include; additional buffering agents;co-solvents; antioxidants including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g., Zn-proteincomplexes); biodegradable polymers such as polyesters; preservatives;and/or salt-forming counterions such as sodium.

α4β7 Antibodies

Anti-α4β7 antibodies suitable for use in the formulations includeantibodies from any desired source, such as fully human antibodies,murine antibodies, rabbit antibodies and the like, and any desiredengineered antibodies, such as chimeric antibodies, humanizedantibodies, and the like. Antigen-binding fragments of any of thesetypes of antibodies, such as Fab, Fv, scFv, Fab′ and F(ab′)₂ fragments,are also suitable for use in the formulations.

The anti-α4β7 antibody can bind to an epitope on the α4 chain (e.g.,humanized MAb 21.6 (Bendig et al., U.S. Pat. No. 5,840,299), on the β7chain (e.g., FIB504 or a humanized derivative (e.g., Fong et al., U.S.Pat. No. 7,528,236)), or to a combinatorial epitope formed by theassociation of the α4 chain with the β7 chain. In one aspect, theantibody binds a combinatorial epitope on the α4β7 complex, but does notbind an epitope on the α4 chain or the β7 chain unless the chains are inassociation with each other. The association of α4 integrin with β7integrin can create a combinatorial epitope for example, by bringinginto proximity residues present on both chains which together comprisethe epitope or by conformationally exposing on one chain, e.g., the α4integrin chain or the β7 integrin chain, an epitopic binding site thatis inaccessible to antibody binding in the absence of the properintegrin partner or in the absence of integrin activation. In anotheraspect, the anti-α4β7 antibody binds both the α4 integrin chain and theβ7 integrin chain, and thus, is specific for the α4β7 integrin complex.Such antibodies can bind α4β7 but not bind α4β1, and/or not bindα_(E)β7, for example. In another aspect, the anti-α4β7 antibody binds tothe same or substantially the same epitope as the Act-1 antibody(Lazarovits, A. I. et al., J. Immunol., 133(4): 1857-1862 (1984),Schweighoffer et al., J. Immunol., 151(2): 717-729, 1993; Bednarczyk etal., J. Biol. Chem., 269(11): 8348-8354, 1994). Murine ACT-1 Hybridomacell line, which produces the murine Act-1 monoclonal antibody, wasdeposited under the provisions of the Budapest Treaty on Aug. 22, 2001,on behalf Millennium Pharmaceuticals, Inc., 40 Landsdowne Street,Cambridge, Mass. 02139, U.S.A., at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., underAccession No. PTA-3663. In another aspect, the anti-α4β7 antibody is ahuman antibody or an α4β7 binding protein using the CDRs provided inU.S. Patent Application Publication No. 2010/0254975.

In one aspect, the anti-α4β7 antibody inhibits binding of α4β7 to one ormore of its ligands (e.g. the mucosal addressin, e.g., MAdCAM (e.g.,MAdCAM-1), fibronectin, and/or vascular addressin (VCAM)). PrimateMAdCAMs are described in the PCT publication WO 96/24673, the entireteachings of which are incorporated herein by this reference. In anotheraspect, the anti-α4β7 antibody inhibits binding of α4β7 to MAdCAM (e.g.,MAdCAM-1) and/or fibronectin without inhibiting the binding of VCAM.

In one aspect, the anti-α4β7 antibodies for use in the formulations arehumanized versions of the mouse Act-1 antibody. Suitable methods forpreparing humanized antibodies are well-known in the art. Generally, thehumanized anti-α4β7 antibody will contain a heavy chain that containsthe 3 heavy chain complementarity determining regions (CDRs, CDR1, SEQID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) of the mouse Act-1antibody and suitable human heavy chain framework regions; and alsocontain a light chain that contains the 3 light chain CDRs (CDR1, SEQ IDNO:11, CDR2, SEQ ID NO:12 and CDR3, SEQ ID NO:13) of the mouse Act-1antibody and suitable human light chain framework regions. The humanizedAct-1 antibody can contain any suitable human framework regions,including consensus framework regions, with or without amino acidsubstitutions. For example, one or more of the frame work amino acidscan be replaced with another amino acid, such as the amino acid at thecorresponding position in the mouse Act-1 antibody. The human constantregion or portion thereof, if present, can be derived from the κ or λlight chains, and/or the γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g., α1, α2),δ or ε heavy chains of human antibodies, including allelic variants. Aparticular constant region (e.g., IgG1), variant or portions thereof canbe selected in order to tailor effector function. For example, a mutatedconstant region (variant) can be incorporated into a fusion protein tominimize binding to Fc receptors and/or ability to fix complement (seee.g., Winter et al., GB 2,209,757 B; Morrison et al., WO 89/07142;Morgan et al., WO 94/29351, Dec. 22, 1994). Humanized versions of Act-1antibody were described in PCT publications nos. WO98/06248 andWO07/61679, the entire teachings of each of which are incorporatedherein by this reference.

In another aspect, the anti-α4β7 humanized antibodies for use in theformulation comprise a heavy chain variable region comprising aminoacids 20 to 140 of SEQ ID NO:2, and a light chain variable regioncomprising amino acids 20 to 131 of SEQ ID NO:4 or amino acids 21 to 132of SEQ ID NO:5. If desired, a suitable human constant region(s) can bepresent. For example, the humanized anti-α4β7 antibody can comprise aheavy chain that comprises amino acids 20 to 470 of SEQ ID NO:2 and alight chain comprising amino acids 21 to 239 of SEQ ID NO:5. In anotherexample, the humanized anti-α4β7 antibody can comprise a heavy chainthat comprises amino acids 20 to 470 of SEQ ID NO:2 and a light chaincomprising amino acids 20 to 238 of SEQ ID NO:4. FIG. 4 shows analignment which compares the generic light chains of human antibodieswith murine antibodies. The alignment illustrates that the humanizedlight chain of vedolizumab (e.g., Chemical Abstract Service (CAS,American Chemical Society) Registry number 943609-66-3), with two mouseresidues switched for human residues, is more human than the light chainof LDP-02 (FIG. 3). In addition, LDP-02 has the somewhat hydrophobic,flexible alanine 114 and a hydrophilic site (Aspartate 115) that isreplaced in vedolizumab with the slightly hydrophilichydroxyl-containing threonine 114 and hydrophobic, potentially inwardfacing valine 115 residue.

Further substitutions to the antibody sequence can be, for example,mutations to the heavy and light chain framework regions, such as amutation of isoleucine to valine on residue 2 of SEQ ID NO:14; amutation of methionine to valine on residue 4 of SEQ ID NO:14; amutation of alanine to glycine on residue 24 of SEQ ID NO:15; a mutationof arginine to lysine at residue 38 of SEQ ID NO:15; a mutation ofalanine to arginine at residue 40 of SEQ ID NO:15; a mutation ofmethionine to isoleucine on residue 48 of SEQ ID NO:15; a mutation ofisoleucine to leucine on residue 69 of SEQ ID NO:15; a mutation ofarginine to valine on residue 71 of SEQ ID NO:15; a mutation ofthreonine to isoleucine on residue 73 of SEQ ID NO:15; or anycombination thereof; and replacement of the heavy chain CDRs with theCDRs (CDR1, SEQ ID NO:8, CDR2, SEQ ID NO:9 and CDR3, SEQ ID NO:10) ofthe mouse Act-1 antibody; and replacement of the light chain CDRs withthe light chain CDRs (CDR1, SEQ ID NO:11, CDR2, SEQ ID NO:12 and CDR3,SEQ ID NO:13) of the mouse Act-1 antibody.

In some embodiments, the anti-α4β7 humanized antibodies for use in theformulation comprise a heavy chain variable region that has about 95%,96%, 97%, 98%, or 99% sequence identity to amino acids 20 to 140 of SEQID NO:2, and a light chain variable region that has about 95%, 96%, 97%,98%, or 99% sequence identity to amino acids 20 to 131 of SEQ ID NO:4 oramino acids 21 to 132 of SEQ ID NO:5. Amino acid sequence identity canbe determined using a suitable sequence alignment algorithm, such as theLasergene system (DNASTAR, Inc., Madison, Wis.), using the defaultparameters. In an embodiment, the anti-α4β7 antibody for use in theformulation is vedolizumab (CAS, American Chemical Society, Registrynumber 943609-66-3).

Other α4β7 antibodies may also be used in the formulations and dosingregimes described herein. For example, the α4β7 antibodies described inUS 2010/0254975 (Amgen, Inc.), incorporated by reference herein in itsentirety, are suitable for use in the formulations and methods oftreating inflammatory bowel disease in an individual.

The anti-α4β7 antibody can be produced by expression of nucleic acidsequences encoding each chain in living cells, e.g., cells in culture. Avariety of host-expression vector systems may be utilized to express theantibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an anti-α4β7 antibody in situ. These include but arenot limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3, NS0 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Forexample, mammalian cells such as Chinese hamster ovary cells (CHO), inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking et al., Gene 45:101 (1986); Cockett etal., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to Chinese hamster ovary (CHO), NS0,HeLa, VERY, baby hamster kidney (BHK), monkey kidney (COS), MDCK, 293,3T3, WI38, human hepatocellular carcinoma cells (e.g., Hep G2), breastcancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 andT47D, and normal mammary gland cell line such as, for example, CRL7030and Hs578Bst.

The glycosylation machinery of different cell types can produceantibodies with different glycosylation composition than in another celltype, or no glycosylation, as with bacterial cells. In one aspect, celltypes for production of the anti-α4β7 antibody are mammalian cells, suchas NS0 or CHO cells. In one aspect, the mammalian cells can comprise thedeletion of an enzyme involved in cell metabolism and the exogenous geneof interest can be operably linked to a replacement enzyme, e.g., in aconstruct or vector for introduction into the cells, e.g., bytransformation or transfection. The construct or vector with theexogenous gene confers to the cells which host the construct or vector aselection advantage to encourage production of the polypeptide encodedby the exogenous gene. In one embodiment, CHO cells are DG44 cells(Chasin and Urlaub (1980) PNAS USA 77:4216), comprising the deletion orinactivation of the dihydrofolate reductase gene. In another embodiment,CHO cells are CHO K1 cells comprising the deletion or inactivation ofthe glutamine synthase gene (see, e.g., U.S. Pat. No. 5,122,464 or5,827,739).

Solid Formulations

Solid formulations of the invention are generally prepared by drying aliquid formulation. Any suitable method of drying can be used, such aslyophilization or spray drying. Lyophilization involves freezing aliquid formulation, usually in the container that will be used to store,ship and distribute the formulation (e.g., a vial). (See, e.g., Gatlinand Nail in Protein Purification Process Engineering, ed. Roger G.Harrison, Marcel Dekker Inc., 317-367 (1994).) Once the formulation isfrozen, the atmospheric pressure is reduced and the temperature isadjusted to allow removal of the frozen solvent e.g., throughsublimation. This step of the lyophilization process is sometimesreferred to as primary drying. If desired, the temperature can then beraised to remove any solvent that is still bound to the dry formulationby evaporation. This step of the lyophilization process is sometimesreferred to as secondary drying. When the formulation has reached thedesired degree of dryness, the drying process is concluded and thecontainers are sealed. The final solid formulation is sometimes referredto as a “lyophilized formulation” or a “cake.” The lyophilizationprocess can be performed using any suitable equipment. Suitablelyophilization equipment is available from a number of commercialsources (e.g., SP Scientific, Stone Ridge, N.Y.).

A variety of suitable apparatuses can be used to dry liquid formulationsto produce a solid (e.g., lyophilized) formulation. Generally,lyophilized formulations are prepared by those of skill in the art usinga sealed chamber that contains shelves, on which vials of the liquidformulation to be dried are placed. The temperature of the shelves, aswell as cooling and heating rate can be controlled, as can the pressureinside the chamber. It will be understood that various processparameters discussed herein refer to processes performed using this typeof apparatus. Persons of ordinary skill can easily adapt the parametersdescribed herein to other types of drying apparatuses if desired.

Suitable temperatures and the amount of vacuum for primary and secondarydrying can be readily determined by a person of ordinary skill. Ingeneral, the formulation is frozen at a temperature of about −30° C. orless, such as −40° C. or −50° C. The rate of cooling can affect theamount and size of ice crystals in the matrix. Primary drying isgenerally conducted at a temperature that is about 10° C., about 20° C.,about 30° C., about 40° C. or about 50° C. warmer than the freezingtemperature. In one aspect, the primary drying conditions can be set tomaintain the anti-α4β7 antibody below the glass transition temperatureor collapse temperature of the formulation. Above the collapsetemperature, the amorphous frozen matrix can flow (collapse), with aresult that the protein molecules may not be surrounded by a rigid,solid matrix, and the protein molecules may not be stable in thecollapsed matrix. Also, the formulation can be difficult to fully dry ifcollapse occurs. The resulting higher amounts of moisture in theformulation can lead to higher rates of protein degradation and adecrease in the amount of time that the lyophilized product can bestored before its quality diminishes to unacceptable levels. In oneaspect, the shelf temperature and chamber pressure are selected tomaintain the product temperature below the collapse temperature duringprimary drying. The glass transition temperature of a frozen formulationcan be measured by methods known in the art, e.g., by differentialscanning calorimetry (DSC). The collapse temperature can be measured bymethods known in the art, e.g. freeze-drying microscopy. The ratio ofnon-reducing sugar to protein (mole:mole) and the amounts of otherformulation components will impact the glass transition temperature andcollapse temperature. In some embodiments, a glass transitiontemperature for an α4β7 antibody formulation is about −35° C. to about−10° C., about −35° C. to about −25° C., or about −35° C. to about −29°C. In another embodiment, the glass transition temperature of an α4β7antibody formulation is about −29° C. In some embodiments, the glasstransition temperature of an α4β7 antibody formulation is about −30° C.,about −31° C., about −32° C., about −33° C., about −34° C., about −35°C. or about −36° C. In some embodiments, a collapse temperature of anα4β7 antibody formulation is about −30° C. to about 0° C., about −28° C.to about −25° C., or about −20° C. to about −10° C. In anotherembodiment, the collapse temperature of an α4β7 antibody formulation isabout −26° C. Without wishing to be bound by any particular theory, thefaster the ramp-up rate, the higher the collapse temperature of theproduct. The primary drying step can remove at least 50%, at least 60%,at least 70% or more of the solvent. In one aspect, the primary dryingstep removes more than 80% of the solvent from the anti-α4β7 antibodyformulation.

Primary drying is dependent on shelf temperature and pressure. Theconditions for primary drying can be determined empirically withlyophilization under different process parameters. Primary drying mayalso be mathematically modeled based on product temperature. Mass andheat transfer equations (Milton, et al. (1997) PDA J of Pharm Sci &Tech, 51: 7-16), coupled with knowledge of Rp and Kv, allow forunderstanding the combination and interaction of input variablesincluding process input variables such as shelf temperature and pressureand formulation variables which are captured in the Rp value. Thesemodels can aid in determining the parameters to be used for an efficientprocess based on the limitations of the product temperature by thecollapse temperature and equipment capability.

$\begin{matrix}{\frac{\mathbb{d}m}{\mathbb{d}t} = \frac{A_{p\;}( {P_{o} - P_{c}} )}{R_{p}}} & {{Equation}\mspace{14mu} 1} \\{{\ln\; P_{0}} = {{{- 6144.96}/T_{p}} + 24.0185}} & {{Equation}\mspace{14mu} 2} \\{\frac{\mathbb{d}Q}{\mathbb{d}t} = {A_{v}{K_{v}( {T_{s} - T_{p}} )}}} & {{Equation}\mspace{14mu} 3} \\{\frac{\mathbb{d}Q}{\mathbb{d}t} = {\Delta\; H_{s}\frac{\mathbb{d}m}{\mathbb{d}t}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

Equation 1 relates the sublimation rate (dm/dt) during primary drying tothe internal cross-sectional area of the container (A_(p)), the vaporpressure of ice (P_(o)), the pressure of the chamber (P_(c)), and anarea normalized mass transfer resistance for the cake and stopper(R_(p)). P_(o) at the sublimation interface can be determined fromEquation 2, where P_(o) is related to the temperature of the product iceat the sublimation interface, which is an approximation from the producttemperature (T_(p)), which can be measured with a thermocouple at thebottom of the vial or can be derived from the equations above when theother variables are determined. Equation 3 relates the heat transferrate from the shelf to the vials, where A_(v) is the area of the vial,K_(v) is the heat transfer coefficient of the vial, T_(s) is thetemperature of the shelf, and T_(p) is the product temperature. Equation4 couples the heat and mass transfer equations, where ΔH_(s) is the heatof sublimation.

As seen from the equations for primary drying, the shelf temperature(T_(s)), the product temperature (T_(p)), the chamber pressure (P_(c)),the mass transfer resistance of the cake (R_(p)), and the heat transfercoefficient (K_(v)) can affect the sublimation rate.

An optional step after freezing and before primary drying is annealing.In this step the shelf temperature of the lyophilizer is raised abovethe glass transition of the formulation for a short period of time,e.g., about 2 to 6 hours, about 3 to 5 hours, or about 4 hours, then theshelf temperature is lowered again to below the glass transitiontemperature of the formulation. Annealing can be used to crystallizebulking agents and to form larger, more uniform ice crystals. Theannealing process can affect reconstitution time because the annealed,dried cake has a higher surface area than the unannealed, dried cake. Anannealing step of an α4β7 antibody formulation can be at about −30° C.to about −10° C. or about −25° C. to about −15° C. In one aspect, anannealing temperature for an α4β7 antibody formulation is about −20° C.

Secondary drying is generally conducted at a temperature that is abovethe freezing temperature of the liquid formulation. For example,secondary drying can be conducted at about 10° C., about 20° C., about30° C., about 40° C. or about 50° C. In one aspect, the temperature forsecondary drying is ambient temperature, e.g., 20-30° C. The time forsecondary drying should be sufficient to reduce the amount of moistureto <5%.

In another aspect, the lyophilization cycle includes freezing at about−45° C., annealing at about −20° C., refreezing at about −45° C.,primary drying at about −24° C. and 150 mTorr, and secondary drying atabout 27° C. and 150 mTorr.

R_(p) is affected by the solids content of the frozen DP and by the DP'sthermal history (freeze, anneal, and refreeze stages) which affects thepore structure of the cake. The thermal history can also affect thesecondary drying stage, where a larger surface area can aid indesorption of water (Pikal, et al. (1990) Int. J. Pharm., 60: 203-217).Useful process parameters to control during the primary and secondarylyophilization stages can be the shelf temperature and chamber pressureduring each stage of the drying cycle.

For scale-up, freeze dryer load and solid content can affect the dryingcycle. Primary drying time can be affected by the solids content in theformulation. At higher solids contents, e.g., where overall solids(excipients and/or protein) concentrations vary more than 10 w/v % ormore than 15 w/v %, e.g., 50 to 100% variance from formulations whosedrying time is determined, the drying time can be affected. For example,a high solids content formulation can have a longer drying time than alow solids content formulation. In some embodiments, the percent usageof freeze dryer capacity can range from about 25 to about 100%. Athigher loading % of capacity, the primary drying time can increase up to2-fold in comparison to a lower loading % capacity. The differencesbetween the primary drying times at different load % increases as thesolids content increases. In one embodiment, the solids content is lessthan 20-25% and the load is from 25-100%.

Vial size can be selected based on the surface area which will beexposed to the shelf and to the vacuum during lyophilization. Dryingtime is directly proportional to cake height, thus the vial size may bechosen based upon what is determined to be a reasonable cake height. Avial with a large diameter relative to volume can provide a high amountof contact with the shelf for efficient heat transfer during thelyophilization cycle. A dilute antibody solution in a high volume ofliquid will require more time for drying. A balance in vial size versusformulation volume needs to be struck, because larger vials can be moreexpensive to store and ship and have a larger headspace to formulationratio and may expose a high proportion of the formulation to thedegradative effects of moisture during long term storage. For a 300 mgdose, anti-α4β7 antibody formulation can have a volume of 3 ml, 5 ml, 6ml, 10 ml, 20 ml, 50 ml or 100 ml prior to lyophilization. In oneaspect, the vial size is 20 ml for a 60 mg/ml solution in a 300 mg dose.

After lyophilization, the vial can be sealed, e.g., stoppered, under avacuum. Alternatively, a gas, e.g., dry air or nitrogen, can be allowedinto the vial prior to sealing. Where oxidation is a concern, the gasallowed into the lyophilization chamber can comprise a gas which retardsor prevents oxidation of the lyophilized product. In one aspect, the gasis a non-oxygenated gas, e.g., nitrogen, or an inert gas, e.g., helium,neon, argon, krypton or xenon. In another aspect, the gas is nitrogen orargon.

In some embodiments, the pre-lyophilization anti-α4β7 antibodyformulation volume is the same as the pre-administration reconstitutedsolution volume. For example, a formulation which is about 5.5 mlpre-lyophilization can be reconstituted to a volume of about 5.5 ml, byadding an amount of liquid, e.g. water or saline, that takes intoaccount the volume of the dry solids. In other embodiments, it may bedesirable to lyophilize the anti-α4β7 antibody formulation in adifferent volume than the reconstituted solution volume. For example,the anti-α4β7 antibody formulation can be lyophilized as a dilutesolution, e.g. 0.25×, 0.5×, or 0.75× and reconstituted to 1× by addingless liquid, e.g., 75% less, half, or 25% less than thepre-lyophilization volume. In an embodiment, a 300 mg dose can belyophilized as a 30 mg/ml antibody solution in 5% sucrose andreconstituted to a 60 mg/ml antibody solution in 10% sucrose.Alternatively, the lyophilized anti-α4β7 antibody formulation can bereconstituted into a more dilute solution than the pre-lyophilizedformulation.

Treatment with the Antibody Formulation

In one aspect, the invention provides a method of treating a disease ordisorder in a subject comprising administering to a subject theanti-α4β7 antibody formulation described herein in an amount effectiveto treat the disease or disorder, e.g., in humans. The human subject maybe an adult (e.g., 18 years or older), an adolescent, or a child. Thehuman subject may be a person 65 years or older. In contrast toalternative therapeutic dosing regimens, a human subject 65 years orolder does not require any modification of the dosing regimen describedherein, and may be administered the conventional anti-α4β7 antibodyformulation described herein.

The subject may have had a lack of an adequate response with, loss ofresponse to, or was intolerant to treatment with an immunomodulator, aTNF-α antagonist, or combinations thereof. The patient may havepreviously received treatment with at least one corticosteroid (e.g.,prednisone) for the inflammatory bowel disease. An inadequate responseto corticosteroids refers to signs and symptoms of persistently activedisease despite a history of at least one 4-week induction regimen thatincluded a dose equivalent to prednisone 30 mg daily orally for 2 weeksor intravenously for 1 week. A loss of response to corticosteroidsrefers to two failed attempts to taper corticosteroids to below a doseequivalent to prednisone 10 mg daily orally. Intolerance ofcorticosteroids includes a history of Cushing's syndrome,osteopenia/osteoporosis, hyperglycemia, insomnia and/or infection.

An immunomodulator may be, for example, oral azathioprine,6-mercaptopurine, or methotrexate. An inadequate response to animmunomodulator refers to signs and symptoms of persistently activedisease despite a history of at least one 8 week regimen or oralazathioprine (≧1.5 mg/kg), 6-mercaptopurine (≧0.75 mg/kg), ormethotrexate (≧12.5 mg/week). Intolerance of an immunomodulatorincludes, but is not limited to, nausea/vomiting, abdominal pain,pancreatitis, LFT abnormalities, lymphopenia, TPMT genetic mutationand/or infection.

In one aspect, the subject may have had a lack of an adequate responsewith, loss of response to, or was intolerant to treatment a TNF-αantagonist. A TNF-α antagonist is, for example, an agent that inhibitsthe biological activity of TNF-α, and preferably binds TNF-α, such as amonoclonal antibody, e.g., REMICADE (infliximab), HUMIRA (adalimumab),CIMZIA (certolizumab pegol), SIMPONI (golimumab) or a circulatingreceptor fusion protein such as ENBREL (etanercept). An inadequateresponse to a TNF-α antagonist refers to signs and symptoms ofpersistently active disease despite a history of at least one 4 weekinduction regimen of infliximab 5 mg/kg IV, 2 doses at least 2 weeksapart; one 80 mg subcutaneous dose of adalimumab, followed by one 40 mgdose at least two weeks apart; or 400 mg subcutaneously of certolizumabpegol, 2 doses at least 2 weeks apart. A loss of response to a TNF-αantagonist refers to recurrence of symptoms during maintenance dosingfollowing prior clinical benefit. Intolerance of a TNF-α antagonistincludes, but is not limited to infusion related reaction,demyelination, congestive heart failure, and/or infection.

A loss of maintenance of remission, as used herein for ulcerativecolitis subjects, refers to an increase in Mayo score of at least 3points and a Modified Baron Score of at least 2.

In another aspect, the present invention provides anti-α4β7 antibodyformulations which (1) can bind α4β7 integrin in vitro and/or in vivo;and (2) can modulate an activity or function of an α4β7 integrin, suchas (a) binding function (e.g., the ability of α4β7 integrin to bind toMAdCAM (e.g., MAdCAM-1), fibronectin and/or VCAM-1) and/or (b) leukocyteinfiltration function, including recruitment and/or accumulation ofleukocytes in tissues (e.g., the ability to inhibit lymphocyte migrationto intestinal mucosal tissue). In one embodiment, an antibody in theformulation can bind an α4β7 integrin, and can inhibit binding of theα4β7 integrin to one or more of its ligands (e.g., MAdCAM (e.g.,MAdCAM-1), VCAM-1, fibronectin), thereby inhibiting leukocyteinfiltration of tissues (including recruitment and/or accumulation ofleukocytes in tissues). In another embodiment, an antibody in theformulation can bind an α4β7 integrin, and can selectively inhibitbinding of the α4β7 integrin to one or more of its ligands (e.g., MAdCAM(e.g., MAdCAM-1), VCAM-1, fibronectin), thereby inhibiting leukocyteinfiltration of tissues (including recruitment and/or accumulation ofleukocytes in tissues). Such anti-α4β7 antibody formulations can inhibitcellular adhesion of cells bearing an α4β7 integrin to vascularendothelial cells in mucosal tissues, including gut-associated tissues,lymphoid organs or leukocytes (especially lymphocytes such as T or Bcells) in vitro and/or in vivo. In yet another embodiment, the anti-α4β7antibody formulation of the present invention can inhibit theinteraction of α4β7 with MAdCAM (e.g., MAdCAM-1) and/or fibronectin. Instill yet another embodiment, the anti-α4β7 antibody formulation of thepresent invention can inhibit the interaction of α4β7 with MAdCAM (e.g.,MAdCAM-1) and/or fibronectin selectively, e.g., without inhibiting theinteraction of α4β7 with VCAM.

The anti-α4β7 antibody formulations of the present invention can be usedto modulate (e.g., inhibit (reduce or prevent)) binding function and/orleukocyte (e.g., lymphocyte, monocyte) infiltration function of α4β7integrin. For example, humanized immunoglobulins which inhibit thebinding of α4β7 integrin to a ligand (i.e., one or more ligands) can beadministered according to the method in the treatment of diseasesassociated with leukocyte (e.g., lymphocyte, monocyte) infiltration oftissues (including recruitment and/or accumulation of leukocytes intissues), particularly of tissues which express the molecule MAdCAM(e.g., MAdCAM-1).

An effective amount of an anti-α4β7 antibody formulation of the presentinvention (i.e., one or more) is administered to an individual (e.g., amammal, such as a human or other primate) in order to treat such adisease. For example, inflammatory diseases, including diseases whichare associated with leukocyte infiltration of the gastrointestinal tract(including gut-associated endothelium), other mucosal tissues, ortissues expressing the molecule MAdCAM (e.g., MAdCAM-1) (e.g.,gut-associated tissues, such as venules of the lamina propria of thesmall and large intestine; and mammary gland (e.g., lactating mammarygland)), can be treated according to the present method. Similarly, anindividual having a disease associated with leukocyte infiltration oftissues as a result of binding of leukocytes to cells (e.g., endothelialcells) expressing MAdCAM (e.g., MAdCAM-1) can be treated according tothe present invention.

In one embodiment, diseases which can be treated accordingly includeinflammatory bowel disease (IBD), such as ulcerative colitis, Crohn'sdisease, ileitis, Celiac disease, nontropical Sprue, enteropathyassociated with seronegative arthropathies, microscopic or collagenouscolitis, eosinophilic gastroenteritis, or pouchitis resulting afterproctocolectomy, and ileoanal anastomosis. Preferably, the inflammatorybowel disease is Crohn's disease or ulcerative colitis. The ulcerativecolitis may be moderate to severely active ulcerative colitis. Treatmentmay result in mucosal healing in patients suffering from moderate toseverely active ulcerative colitis. Treatment may also result in areduction, elimination, or reduction and elimination of corticosteroiduse by the patient.

Pancreatitis and insulin-dependent diabetes mellitus are other diseaseswhich can be treated using the formulations of the invention. It hasbeen reported that MAdCAM (e.g., MAdCAM-1) is expressed by some vesselsin the exocrine pancreas from NOD (nonobese diabetic) mice, as well asfrom BALB/c and SJL mice. Expression of MAdCAM-1 was reportedly inducedon endothelium in inflamed islets of the pancreas of the NOD mouse, andMAdCAM-1 was the predominant addressin expressed by NOD isletendothelium at early stages of insulitis (Hanninen, A., et al., J. Clin.Invest., 92: 2509-2515 (1993)). Treatment of NOD mice with eitheranti-MAdCAM (e.g., anti-MAdCAM-1) or anti β7 antibodies prevented thedevelopment of diabetes (Yang et al., Diabetes, 46:1542-1547 (1997)).Further, accumulation of lymphocytes expressing α4β7 within islets wasobserved, and MAdCAM-1 was implicated in the binding of lymphoma cellsvia α4β7 to vessels from inflamed islets (Hanninen, A., et al., J. Clin.Invest., 92: 2509-2515 (1993)) or to the gastrointestinal tract inmantle cell lymphoma (Geissmann et al., Am. J. Pathol., 153:1701-1705(1998)).

Examples of inflammatory diseases associated with mucosal tissues whichcan be treated using a formulation of the invention includecholecystitis, cholangitis (Adams and Eksteen Nature Reviews 6:244-251(2006) Grant et al., Hepatology 33:1065-1072 (2001)), e.g., primarysclerosing cholangitis, Behcet's disease, e.g., of the intestine, orpericholangitis (bile duct and surrounding tissue of the liver), andgraft versus host disease (e.g., in the gastrointestinal tract (e.g.,after a bone marrow transplant) (Petrovic et al. Blood 103:1542-1547(2004)). As seen in Crohn's disease, inflammation often extends beyondthe mucosal surface, accordingly chronic inflammatory diseases, such assarcoidosis, chronic gastritis, e.g., autoimmune gastritis (Katakai etal., Int. Immunol., 14:167-175 (2002)) and other idiopathic conditionscan be amenable to treatment.

The invention also relates to a method of inhibiting leukocyteinfiltration of mucosal tissue. The invention also relates to a methodfor treating cancer (e.g., an α4β7 positive tumor, such as a lymphoma).Other examples of inflammatory diseases associated with mucosal tissueswhich can be treated using a formulation of the invention includemastitis (mammary gland) and irritable bowel syndrome.

Diseases or pathogens whose etiologies exploit the interaction of MAdCAM(e.g., MAdCAM-1) with α4β7 can be treated with an anti-α4β7 antibody ina formulation described herein. Examples of such diseases includeimmunodeficiency disorders, such as caused by human immunodeficiencyvirus (see e.g., WO2008140602).

A formulation of the invention is administered in an effective amountwhich inhibits binding of α4β7 integrin to a ligand thereof. Fortherapy, an effective amount will be sufficient to achieve the desiredtherapeutic (including prophylactic) effect (such as an amountsufficient to reduce or prevent α4β7 integrin-mediated binding and/orsignaling, thereby inhibiting leukocyte adhesion and infiltration and/orassociated cellular responses). An effective amount of an anti-α4β7antibody, e.g., an effective titer sufficient to maintain saturation,e.g., neutralization, of α4β7 integrin, can induce clinical response orremission in inflammatory bowel disease. A formulation of the inventioncan be administered in a unit dose or multiple doses. The dosage can bedetermined by methods known in the art and can be dependent, forexample, upon the individual's age, sensitivity, tolerance and overallwell-being. Examples of modes of administration include topical routessuch as nasal or inhalational or transdermal administration, enteralroutes, such as through a feeding tube or suppository, and parenteralroutes, such as intravenous, intramuscular, subcutaneous, intraarterial,intraperitoneal, or intravitreal administration. Suitable dosages forantibodies can be from about 0.1 mg/kg body weight to about 10.0 mg/kgbody weight per treatment, for example about 2 mg/kg to about 7 mg/kg,about 3 mg/kg to about 6 mg/kg, or about 3.5 to about 5 mg/kg. Inparticular embodiments, the dose administered is about 0.3 mg/kg, about0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg,about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9mg/kg, or about 10 mg/kg.

The final dosage form, e.g., after dilution of the reconstitutedantibody (e.g., in a saline or 5% dextrose infusion system) of theanti-α4β7 antibody can be about 0.5 mg/ml to about 5 mg/ml foradministration. The final dosage form may be at a concentration ofbetween about 1.0 mg/ml to about 1.4 mg/ml, about 1.0 mg/ml to about 1.3mg/ml, about 1.0 mg/ml to about 1.2 mg/ml, about 1.0 to about 1.1 mg/ml,about 1.1 mg/ml to about 1.4 mg/ml, about 1.1 mg/ml to about 1.3 mg/ml,about 1.1 mg/ml to about 1.2 mg/ml, about 1.2 mg/ml to about 1.4 mg/ml,about 1.2 mg/ml to about 1.3 mg/ml, or about 1.3 mg/ml to about 1.4mg/ml. The final dosage form may be at a concentration of about 0.6mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.1 mg/ml, about 1.2 mg/ml, about 1.3mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.8mg/ml or about 2.0 mg/ml. In one embodiment, the total dose is 180 mg.In another embodiment, the total dose is 300 mg. A 300 mg anti-α4β7antibody dose can be diluted into a 250 ml saline or 5% dextrosesolution for administration.

In some aspects, the dosing regimen has two phases, an induction phaseand a maintenance phase. In the induction phase, the antibody orantigen-binding fragment thereof is administered in a way that quicklyprovides an effective amount of the antibody or antigen binding fragmentthereof suitable for certain purposes, such as inducing immune toleranceto the antibody or antigen-binding fragment thereof or for inducing aclinical response and ameliorating inflammatory bowel disease symptoms.A patient can be administered an induction phase treatment when firstbeing treated by an anti-α4β7 antibody, when being treated after a longabsence from therapy, e.g., more than three months, more than fourmonths, more than six months, more than nine months, more than one year,more than eighteen months or more than two years since anti-α4β7antibody therapy or during maintenance phase of anti-α4β7 antibodytherapy if there has been a return of inflammatory bowel diseasesymptoms, e.g., a relapse from remission of disease. In someembodiments, the induction phase regimen results in a higher mean troughserum concentration, e.g., the concentration just before the next dose,than the mean steady state trough serum concentration maintained duringthe maintenance regimen.

In the maintenance phase, the antibody or antigen-binding fragmentthereof is administered in a way that continues the response achieved byinduction therapy with a stable level of antibody or antigen-bindingfragment thereof. A maintenance regimen can prevent return of symptomsor relapse of inflammatory bowel disease. A maintenance regimen canprovide convenience to the patient, e.g., be a simple dosing regimen orrequire infrequent trips for treatment. In some embodiments, themaintenance regimen can include administration of the anti-α4β7 antibodyor antigen-binding fragment thereof, e.g., in a formulation describedherein, by a strategy selected from the group consisting of low dose,infrequent administration, self-administration and a combination any ofthe foregoing.

In one embodiment, e.g., during an induction phase of therapy, thedosing regimen provides an effective amount of an anti-α4β7 antibody orantigen-binding fragment in a formulation described herein for inducingremission of an inflammatory bowel disease in a human patient. In someembodiments, the effective amount of the anti-α4β7 antibody issufficient to achieve about 5 μg/ml to about 60 μg/ml, about 15 μg/ml toabout 45 μg/ml, about 20 μg/ml to about 30 μg/ml, or about 25 μg/ml toabout 35 μg/ml mean trough serum concentration of the anti-α4β7 antibodyby the end of the induction phase. The duration of induction phase canbe about four weeks, about five weeks, about six weeks, about sevenweeks, or about eight weeks of treatment. In some embodiments, theinduction regimen can utilize a strategy selected from the groupconsisting of high dose, frequent administration, and a combination ofhigh dose and frequent administration of the anti-α4β7 antibody orantigen-binding fragment thereof, e.g., in a formulation describedherein. Induction dosing can be once, or a plurality of more than onedose, e.g., at least two doses. During induction phase, a dose can beadministered once per day, every other day, twice per week, once perweek, once every ten days, once every two weeks or once every threeweeks. In some embodiments, the induction doses are administered withinthe first two weeks of therapy with the anti-α4β7 antibody. In oneembodiment, induction dosing can be once at initiation of treatment (day0) and once at about two weeks after initiation of treatment. In anotherembodiment, the induction phase duration is six weeks. In anotherembodiment, the induction phase duration is six weeks and a plurality ofinduction doses are administered during the first two weeks.

In some embodiments, e.g., when initiating treatment of a patient withsevere inflammatory bowel disease (e.g., in patients who have failedanti-TNFα therapy), the induction phase needs to have a longer durationthan for patients with mild or moderate disease. In some embodiments,the induction phase for a patient with a severe disease can have aduration of at least 6 weeks, at least 8 weeks, at least 10 weeks, atleast 12 weeks or at least 14 weeks. In one embodiment, an inductiondosing regimen for a patient with a severe disease can include a dose atweek 0 (initiation of treatment), a dose at week 2 and a dose at week 6.In another embodiment, an induction dosing regimen for a patient with asevere disease can comprise a dose at week 0 (initiation of treatment),a dose at week 2, a dose at week 6 and a dose at week 10.

In one embodiment, e.g., during a maintenance phase of therapy, thedosing regimen maintains a mean steady state trough serum concentration,e.g., the plateau concentration just before the next dose, of about 5 toabout 25 μg/mL, about 7 to about 20 μg/mL, about 5 to about 10 μg/mL,about 10 to about 20 μg/mL, about 15 to about 25 μg/mL or about 9 toabout 13 μg/mL of anti-α4β7 antibody. In another embodiment, the dosingregimen e.g., during a maintenance phase of therapy, maintains a meansteady state trough serum concentration of about 20 to about 30 μg/mL,about 20 to about 55 μg/mL, about 30 to about 45 μg/mL, about 45 toabout 55 μg/mL or about 35 to about 40 μg/mL of anti-α4β7 antibody.

The dose can be administered once per week, once every 2 weeks, onceevery 3 weeks, once every 4 weeks, once every 6 weeks, once every 8weeks or once every 10 weeks. A higher or more frequent dose, e.g., onceper week, once every 2 weeks, once every 3 weeks or once every 4 weekscan be useful for inducing remission of active disease or for treating anew patient, e.g., for inducing tolerance to the anti-α4β7 antibody. Aless frequent dose, e.g., once every 4 weeks, once every 5 weeks, onceevery 6 weeks, once every 8 weeks or once every 10 weeks, can be usefulfor preventative therapy, e.g., to maintain remission of a patient withchronic disease. In one aspect, the treatment regimen is treatment atday 0, about week 2, about week 6 and every 4 or 8 weeks thereafter. Inone embodiment, the maintenance regimen includes a dose every 8 weeks.In an embodiment, wherein a patient on a one dose every eight weeksmaintenance regimen experiences a return of one or more diseasesymptoms, e.g., has a relapse, the dosing frequency can be increased,e.g., to once every 4 weeks.

The dose can be administered to the patient in about 20 minutes, about25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes.

The dosing regimen can be optimized to induce a clinical response andclinical remission in the inflammatory bowel disease of the patient. Insome embodiments, the dosing regimen does not alter the ratio of CD4 toCD8 in cerebrospinal fluid of patients receiving treatment.

In some aspects, a durable clinical remission, for example, a clinicalremission which is sustained through at least two, at least three, atleast four visits with a caretaking physician within a six month or oneyear period after beginning treatment, may be achieved with an optimizeddosing regimen.

In some aspects, a durable clinical response, for example, a clinicalresponse which is sustained for at least 6 months, at least 9 months, atleast a year, after the start of treatment, may be achieved with anoptimized dosing regimen.

In one embodiment, the dosing regimen comprises an initial dose of 300mg, a second subsequent dose of 300 mg about two weeks after the initialdose, a third subsequent dose of 300 mg at about six weeks after theinitial dose, followed by a fourth and subsequent doses of 300 mg everyfour weeks or every eight weeks after the third subsequent dose.

In some embodiments, the method of treatment, dose or dosing regimenreduces the likelihood that a patient will develop a HAHA response tothe anti-α4β7 antibody. The development of HAHA, e.g., as measured byantibodies reactive to the anti-α4β7 antibody, can increase theclearance of the anti-α4β7 antibody, e.g., reduce the serumconcentration of the anti-α4β7 antibody, e.g., lowering the number ofanti-α4β7 antibody bound to α4β7 integrin, thus making the treatmentless effective. In some embodiments, to prevent HAHA, the patient can betreated with an induction regimen followed by a maintenance regimen. Insome embodiments, there is no break between the induction regimen andthe maintenance regimen. In some embodiments, the induction regimencomprises administering a plurality of doses of anti-α4β7 antibody tothe patient. To prevent HAHA, the patient can be treated with a highinitial dose, e.g., at least 1.5 mg/kg, at least 2 mg/kg, at least 2.5mg/kg, at least 3 mg/kg, at least 5 mg/kg, at least 8 mg/kg, at least 10mg/kg or about 2 to about 6 mg/kg, or frequent initial administrations,e.g., about once per week, about once every two weeks or about onceevery three weeks, of the standard dose when beginning therapy with ananti-α4β7 antibody. In some embodiments, the method of treatmentmaintains at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or at least 95% of patients asHAHA-negative. In other embodiments, the method of treatment maintainspatients as HAHA-negative for at least 6 weeks, at least 10 weeks atleast 15 weeks, at least six months, at least 1 year, at least 2 years,or for the duration of therapy. In some embodiments, the patients, or atleast 30%, at least 40%, at least 50% or at least 60% of patients whodevelop HAHA maintain a low titer, e.g., ≦125, of anti-α4β7 antibody. Inan embodiment, the method of treatment maintains at least 70% ofpatients as HAHA-negative for at least 12 weeks after beginning therapywith an anti-α4β7 antibody.

The formulation may be administered to an individual (e.g., a human)alone or in conjunction with another agent. A formulation of theinvention can be administered before, along with or subsequent toadministration of the additional agent. In one embodiment, more than oneformulation which inhibits the binding of α4β7 integrin to its ligandsis administered. In such an embodiment, an agent, e.g., a monoclonalantibody, such as an anti-MAdCAM (e.g., anti-MAdCAM-1) or an anti-VCAM-1monoclonal antibody can be administered. In another embodiment, theadditional agent inhibits the binding of leukocytes to an endothelialligand in a pathway different from the α4β7 pathway. Such an agent caninhibit the binding, e.g. of chemokine (C-C motif) receptor 9(CCR9)-expressing lymphocytes to thymus expressed chemokine (TECK orCCL25) or an agent which prevents the binding of LFA-1 to intercellularadhesion molecule (ICAM). For example, an anti-TECK or anti-CCR9antibody or a small molecule CCR9 inhibitor, such as inhibitorsdisclosed in PCT publication WO03/099773 or WO04/046092, or anti-ICAM-1antibody or an oligonucleotide which prevents expression of ICAM, isadministered in addition to a formulation of the present invention. Inyet another embodiment, an additional active ingredient (e.g., ananti-inflammatory compound, such as sulfasalazine, azathioprine,6-mercaptopurine, 5-aminosalicylic acid containing anti-inflammatories,another non-steroidal anti-inflammatory compound, a steroidalanti-inflammatory compound, or antibiotics commonly administered forcontrol of IBD (e.g. ciprofloxacin, metronidazole), or another biologicagent (e.g. TNF alpha antagonists) can be administered in conjunctionwith a formulation of the present invention.

In an embodiment, the dose of the co-administered medication can bedecreased over time during the period of treatment by the formulationcomprising the anti-α4β7 antibody. For example, a patient being treatedwith a steroid (e.g. prednisone, prednisolone) at the beginning, orprior to, treating with the anti-α4β7 antibody formulation would undergoa regimen of decreasing doses of steroid beginning as early as 6 weeksof treatment with the anti-α4β7 antibody formulation. The steroid dosewill be reduced by about 25% within 4-8 weeks of initiating tapering, by50% at about 8-12 weeks and 75% at about 12-16 weeks of tapering duringtreatment with the anti-α4β7 antibody formulation. In one aspect, byabout 16-24 weeks of treatment with the anti-α4β7 antibody formulation,the steroid dose can be eliminated. In another example, a patient beingtreated with an anti-inflammatory compound, such as 6-mercaptopurine atthe beginning, or prior to, treating with the anti-α4β7 antibodyformulation would undergo a regimen of decreasing doses ofanti-inflammatory compound similar to the tapering regimen for steroiddosing as noted above.

In one embodiment, the method comprises administering an effectiveamount of a formulation of the invention to a patient. If theformulation is in a solid, e.g., dry state, the process ofadministration can comprise a step of converting the formulation to aliquid state. In one aspect, a dry formulation can be reconstituted,e.g., by a liquid as described above, for use in injection, e.g.intravenous, intramuscular or subcutaneous injection. In another aspect,a solid or dry formulation can be administered topically, e.g., in apatch, cream, aerosol or suppository.

The invention also relates to a method for treating a disease associatedwith leukocyte infiltration of tissues expressing the molecule MAdCAM(e.g., MAdCAM-1). The method comprises administering to a patient inneed thereof an effective amount of an anti-α4β7 antibody formulation ofthe invention. In an embodiment, the disease is graft versus hostdisease. In some embodiments, the disease is a disease associated withleukocyte infiltration of tissues as a result of binding of leukocytesexpressing α4β7 integrin to gut-associated endothelium expressing themolecule MAdCAM (e.g., MAdCAM-1). In other embodiments, the disease isgastritis (e.g., eosinophilic gastritis or autoimmune gastritis),pancreatitis, or insulin-dependent diabetes mellitus. In yet otherembodiments, the disease is cholecystitis, cholangitis, orpericholangitis.

The invention also relates to a method for treating inflammatory boweldisease in a patient. In one embodiment, the method comprisesadministering to the patient an effective amount of an anti-α4β7antibody formulation of the invention. In some embodiments, theinflammatory bowel disease is ulcerative colitis or Crohn's disease. Inother embodiments, the inflammatory bowel disease is Celiac disease,enteropathy associated with seronegative arthropathies, microscopic orcollagenous colitis, gastroenteritis (e.g., eosinophilicgastroenteritis), or pouchitis.

In some embodiments, treatment with an anti-α4β7 antibody does not alterthe ratio of CD4:CD8 lymphocytes. CD4:CD8 ratios can be measured inblood, lymph node aspirate, and cerebro-spinal fluid (CSF). The CSFCD4+:CD8+ lymphocyte ratios in healthy individuals are typically greaterthan or equal to about 1. (Svenningsson et al., J. Neuroimmunol. 1995;63:39-46; Svenningsson et al., Ann Neurol. 1993; 34:155-161). Animmunomodulator can alter the CD4:CD8 ratio to less than 1.

Articles of Manufacture

In another aspect, the invention is an article of manufacture whichcontains the pharmaceutical formulation of the present invention andprovides instructions for its use. The article of manufacture comprisesa container. Suitable containers include, for example, bottles, vials(e.g., dual chamber vials, a vial of liquid formulation with or withouta needle, a vial of solid formulation with or without a vial ofreconstitution liquid with or without a needle), syringes (such as dualchamber syringes, preloaded syringes) and test tubes. The container maybe formed from a variety of materials such as glass, metal or plastic.The container holds the formulation and a label on, or associated with,the container may indicate directions for use. In another embodiment,the formulation can be prepared for self-administration and/or containinstructions for self-administration. In one aspect, the containerholding the formulation may be a single-use vial. In another aspect, thecontainer holding the formulation may be a multi-use vial, which allowsfor repeat administration (e.g., from 2-6 administrations) of theformulation, e.g., using more than one portion of a reconstitutedformulation. The article of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes and package insertswith instructions for use as noted in the previous section.

Clinical and Quality Analysis

In another aspect, the invention is a method for determining that apharmaceutical formulation meets product quality standards. The methodmay comprise evaluation of a lyophilized pharmaceutical formulation(e.g., humanized anti-α4β7 antibody) comprising inspecting theformulation to assess appearance, determining reconstitution time,determining moisture content of lyophilized formulation, measuringaggregates in lyophilized formulation, measuring fragmentation,measuring oxidation/deamidation, and optionally assessing biologicalactivity and potency, wherein achievement of pre-determined standardsdemonstrates product is indicated for clinical use.

Acceptable quality levels include ≦5.0% moisture, ≦40 minutesreconstitution time, pH 6.3±0.3 of reconstituted liquid, 54.0 to 66.0mg/ml antibody concentration, ≧55.0% major isoform by CEX, ≧96.0%monomer by SEC, ≦2.5% high molecular weight (aggregates), ≧90% H+Lchains by SDS-PAGE, 60-140% of the reference standard adhesion.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areincorporated herein by reference.

Development Protocol for Making Formulation

A. Anti-α4β7 Antibody Solution

Bottles of frozen, high concentration anti-α4β7 antibody preparation(vedolizumab, 50 mM histidine, 125 mM arginine, 0.06% polysorbate 80, pH6.3) are thawed at room temperature for 16-24 hours. Thawed bottles arepooled into a stainless steel compounding vessel and mixed. Thepreparation is then diluted with Dilution Buffer A (50 mM histidine, 125mM arginine, 0.06% polysorbate 80, pH 6.3) to 80 mg/mL of vedolizumaband mixed. Sucrose is then added by diluting the preparation withDilution Buffer B which contains sucrose (50 mM histidine, 125 mMarginine, 40% sucrose, 0.06% polysorbate 80, pH 6.3). This step dilutesthe anti-α4β7 antibody preparation to a liquid formulation of 60 mg/mLvedolizumab, 50 mM histidine, 125 mM arginine, 10% sucrose, 0.06%polysorbate 80, pH 6.3.

B. Lyophilization

Anti-α4β7 antibody liquid formulation at 60 mg/ml in 50 mM histidine,125 mM arginine, 0.06% polysorbate 80, 10% sucrose, at pH6.3 is filledinto 20 mL glass vials with 5.52 mL per vial and the stoppers are placedin the lyophilization position. Vials are loaded onto shelves set atabout 20° C. in a lyophilizer. After loading all vials and closing thedoor, the shelf temperature is lowered to freeze the solution, about−45° C. After 3 hours at this temperature, the temperature of theshelves is raised to −20° C. for annealing. After annealing for fourhours, the temperature of the shelves is lowered to re-freeze thesolution, about −45° C. After equilibration of the vials to thistemperature, the air is evacuated from the chamber. When the pressure is150 mTorr, the shelf temperature is ramped to the primary dryingtemperature, about −24° C. Primary drying proceeds until the all of thecrystalline ice has sublimed from the vials. Then the shelf temperatureis raised to 27° C. for secondary drying for 16 hours, until themoisture is approximately less than 2.5% of the lyophilized formulation.When secondary drying is complete, nitrogen gas is backfilled into thechamber until ambient pressure is reached. The vials are stoppered andremoved from the lyophilizer.

C. Storage and Use of Lyophilized Anti-α4β7 Antibody

Lyophilized vials of anti-α4β7 antibody are stored at −70° C., −20° C.,2-8° C. or 25° C. for desired periods of time. When ready for use, avial is equilibrated to room temperature. Then the contents of the vialare reconstituted with a syringe containing water for injection (“WFI”)using a 21 G needle. The amount of WFI is determined so the final volumeof the reconstituted antibody solution is the same volume of thepre-lyophilized solution. For a 5.52 ml pre-lyophilization volume, 4.8ml of WFI is added. The vial is gently swirled and then held for 10-30minutes to allow the formulation to reconstitute, then the antibodysolution is removed using a syringe and is added and added to an IV bagfor IV infusion to a patient.

EXEMPLIFICATION Example 1

Comparative Data for Varying % Sugar and Amino Acids in LyophilizedFormulations

A design of experiments approach was performed to look at the effect ofvarying the molar ratio of sugar (sucrose and mannitol) to protein, themolar ratio of arginine to protein, and the molar amount of histidinebuffer. Histidine and arginine are known not to crystallize during thelyophilization process, making them potential cryo or lyo protectants.1.5 mL of formulation was filled into 5 mL vials lyophilized withPrimary Drying at −30° C., 150 mT and Secondary Drying at 20° C., 150mT. The stability of the lyophilized formulations reconstituted to 1.5ml after different storage conditions is shown in Tables 1-3 (compiling60 mg/ml results from two experiments). FIG. 6A shows the predictivemodels for changes in Percent Monomer, Percent Aggregates, and PercentMajor Isoform when stored at 40° C. when pH and the molar ratio of sugarand arginine was varied. The stability of the formulation was best atlow pH and high molar ratio of (sugar+arginine) to protein. At thehistidine molar amounts examined, histidine did not affect the stabilityof the formulation. All formulations had 1-2% moisture during storage.

TABLE 1 Change in Percent Monomer when stored at 5° C., 25° C./60% RH,and 40° C./75% RH for 3 months. Percent Monomer was measured using SizeExclusion Chromatography (SEC). % Monomer by SEC 25° C. 40° C. 60% 75%Formulation 5° C. RH RH 60 mg/mL vedolizumab + t = 0 3 mo 3 mo 3 mo 25mM histidine, 75 mM arginine, 98.1 98.1 97.8 96.5 2% sucrose, 0.05%polysorbate 80, pH 6.3 25 mM histidine, 75 mM arginine, 98.0 98.2 98.097.5 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mMarginine, 98.0 98.3 98.1 97.4 2% sucrose, 0.05% polysorbate 80, pH 6.750 mM histidine, 125 mM arginine, 98.0 98.3 98.1 97.4 4% sucrose, 0.05%polysorbate 80, pH 6.9 50 mM histidine, 125 mM arginine, 98.7 98.4 98.498.1 6% sucrose, 1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mMhistidine, 125 mM arginine, 98.7 98.3 98.1 98.3 9% sucrose, 0.06%polysorbate 80, pH 6.3

TABLE 2 Change in Percent Aggregates when stored 5° C., 25° C./60% RH,and 40° C./75% RH for 3 months. Percent Monomer was measured using SizeExclusion Chromatography (SEC). % Aggregates by SEC 5° C. 25° C. 40° C.Formulation 60 mg/mL vedolizumab + t = 0 3 mo 60% RH 3 mo 75% RH 3 mo 25mM histidine, 75 mM arginine, 2% sucrose, 0.42 0.53 0.89 1.99 0.05%polysorbate 80, pH 6.3 25 mM histidine, 75 mM arginine, 4% sucrose, 0.410.51 0.62 1.15 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mMarginine, 2% sucrose, 0.42 0.47 0.60 1.23 0.05% polysorbate 80, pH 6.750 mM histidine, 125 mM arginine, 4% sucrose, 0.36 0.44 0.52 0.82 0.05%polysorbate 80, pH 6.9 50 mM histidine, 125 mM arginine, 6% sucrose,0.53 0.49 0.51 0.56 1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mMhistidine, 125 mM arginine, 9% sucrose, 0.51 0.51 0.59 0.56 0.06%polysorbate 80, pH 6.3

TABLE 3 Change in Percent Major Isoform when stored at 5° C., 25° C./60%RH, and 40° C./75% RH for 3 months. Major Isoform was measured usingCation Exchange Chromatography (CEX). % Major Isoform by CEX 25° C. 40°C. Formulation 5° C. 60% RH 75% 60 mg/mL vedolizumab + t = 0 3 mo 3 moRH 3 mo 25 mM histidine, 75 mM arginine, 70.5 68.8 67.4 66.3 2% sucrose,0.05% polysorbate 80, pH 6.3 25 mM histidine, 75 mM arginine, 70.8 98.968.0 67.7 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125mM arginine, 70.5 68.9 67.8 66.5 2% sucrose, 0.05% polysorbate 80, pH6.7 50 mM histidine, 125 mM arginine, 70.6 68.9 68.0 67.4 4% sucrose,0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mM arginine, 69.6 69.569.3 67.4 6% sucrose, 1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mMhistidine, 125 mM arginine, 69.5 69.3 69.2 68.1 9% sucrose, 0.06%polysorbate 80, pH 6.3

FIG. 6A shows the predicted models based on the statistical analysis of40° C. data from Tables 1-3. The model for change in percent monomer permonth at 40° C. by SEC analysis is −3.10+(0.386)*pH+0.000516*((moles ofsugar+moles arginine)/moles of protein)). The model for change inpercent aggregate per month at 40° C. by SEC analysis is2.43−(0.263)*pH−0.000787*((moles of sugar+moles arginine)/moles ofprotein)). The model for change in percent major isoform per month at40° C. by CEX analysis is −2.54+(0.109)*pH−0.00130*((moles ofsugar+moles arginine)/moles of protein)). The center line shows theresults for the predictive models and the outer lines show the 95%confidence limit for the predictive models.

FIG. 6B shows alternative models based on the statistical analysis of40° C. data from Tables 1-3 when the input factors are pH, sugar:proteinmolar ratio, and arginine:protein molar ratio. The model for change inpercent monomer per month at 40° C. by SEC analysis is−3.02+(0.370)*pH+0.000482*((moles of sugar)/(moles ofprotein)+0.000657*((moles of arginine/moles of protein). The model forchange in percent aggregate per month at 40° C. by SEC analysis is2.35−(0.244)*pH−0.000727*((moles of sugar)/(moles ofprotein)−0.00102*((moles of arginine)/(moles of protein)). The model forchange in percent major isoform per month at 40° C. by CEX analysis is−2.92+(0.210)*pH+0.00164*((moles of sugar)/)/(moles ofprotein)−0.000220*((moles of arginine)/(moles of protein)). The centerline shows the results for the predictive models and the outer linesshow the 95% confidence limit for the predictive models.

Example 2

Stability Data

Three primary stability batches of the formulation (Batch A, B, and C)were tested for stability after storage at the prescribed storagecondition (5 and 25° C./60% RH for up to 24 months). All three batchescontain the same liquid formulation that was lyophilized: 60 mg/mLanti-α4β7 antibody, 50 mM histidine, 125 mM arginine, 10% sucrose, 0.06%polysorbate 80, pH 6.3. For Batch A, 3.5 mL of solution was filled into20 mL vials and lyophilized, for Batches B and C, 5.52 mL of solutionwas filled into 20 mL vials and lyophilized.

In a separate study, a single drug formulation of 60 mg/ml anti-α4β7antibody, 50 mM histidine, 125 mM arginine, 10% sucrose, 0.06%polysorbate 80, pH 6.3 was lyophilized in two volumes, 3.5 ml and 9.5ml, respectively, to yield Batches R and S for stability samples, whichwere analyzed over 38 months. Blanks are NT (not tested).

The data (Tables 4-19) showed that the antibody formulations remainedstable when stored for up to 38 months at 5° C. and up to 30 months at25° C./60% RH. All product attributes remained within the specificationsthrough the 38 month time point.

TABLE 4 Change in Percent Monomer by SEC when stored at 5° C. Time(months) Batch A Batch B Batch C Batch R Batch S 0 99.8 99.8 99.8 98.998.8 1 99.8 99.1 99.2 98.8 99.2 3 99.8 99.1 99.1 98.8 98.8 6 99.8 99.899.8 98.9 99.0 9 99.1 99.2 99.2 99.2 99.1 12 99.4 99.0 99.0 98.8 98.9 1599.4 99.1 99.1 18 99.5 99.4 99.4 98.9 98.9 24 99.4 99.2 99.2 99.0 99.030 99.2 99.2 38 99.3 99.3

TABLE 5 Change in Percent Aggregates by SEC when stored at 5° C. Time(months) Batch A Batch B Batch C Batch R Batch S 0 0.1 0.1 0.1 0.2 0.2 10.1 0.2 0.2 0.2 0.1 3 0.1 0.2 0.2 0.2 0.2 6 0.2 0.2 0.2 0.2 0.2 9 0.10.2 0.2 0.2 0.2 12 0.2 0.2 0.2 0.2 0.2 15 0.2 0.2 0.2 18 0.2 0.2 0.2 0.20.2 24 0.2 0.2 0.2 0.2 0.2 30 0.2 0.2 38 0.2 0.2

TABLE 6 Change in Percent Major Isoform by CEX when stored at 5° C. Time(months) Batch A Batch B Batch C Batch R Batch S 0 68.6 69.9 69.5 71.771.6 1 67.5 68.9 68.8 71.2 72.0 3 68.7 68.8 68.7 70.4 70.3 6 67.7 68.268.2 71.9 71.9 9 70.0 68.3 67.8 69.2 69.7 12 67.8 68.3 68.1 70.8 70.9 1566.9 67.5 67.5 18 67.4 67.0 66.7 71.0 70.8 24 68.1 69.6 69.1 71.3 70.930 68.5 68.6 38 73.6 73.1

TABLE 7 Change in Percent Acidic Isoforms by CEX when stored at 5° C.Time (months) Batch A Batch B Batch C Batch R Batch S 0 22.8 20.8 21.420.3 20.6 1 21.9 21.7 22.3 21.6 20.3 3 21.7 22.2 22.8 22.0 22.0 6 22.923.1 23.6 21.1 21.4 9 19.8 22.2 22.9 21.8 21.8 12 22.9 21.3 22.1 21.221.2 15 22.7 22.3 22.8 18 22.8 22.3 22.6 21.1 21.5 24 21.7 22.1 22.920.6 20.7 30 22.8 23.2 38 18.9 19.1

TABLE 8 Change in Percent Basic Isoforms by CEX when stored at 5° C.Time (months) Batch A Batch B Batch C Batch R Batch S 0 8.5 9.3 9.1 8.17.8 1 10.7 9.4 8.9 7.3 7.7 3 9.7 9.0 8.5 7.6 7.8 6 9.5 8.7 8.2 7.0 6.7 910.2 9.6 9.3 9.0 8.4 12 9.3 10.3 9.9 8.0 7.9 15 10.4 10.1 9.7 18 9.810.7 10.7 7.9 7.7 24 10.2 8.3 8.1 8.1 8.3 30 8.7 8.2 38 7.5 7.7

TABLE 9 Change in % (H + L) by Reduced-SDS Page when stored at 5° C.Time (months) Batch A Batch B Batch C Batch R Batch S 0 98 98 98 96 96 198 94 98 98 98 3 98 98 98 98 98 6 98 97 97 97 97 9 97 97 97 98 98 12 9896 97 98 98 15 97 98 97 18 98 97 97 99 99 24 98 98 98 99 99 30 97 97 3899 99

TABLE 10 Change in Binding Efficacy when stored at 5° C. Time (months)Batch A Batch B Batch C Batch R Batch S 0 107 106 105 93 102 1 106 106103 103 111 3 101 109 108 91 98 6 97 106 105 114 121 9 100 93 88 102 10212 103 101 87 119 116 15 105 90 94 18 86 101 96 95 104 24 92 82 95 81101 30 87 94 38 89 91

TABLE 11 Change in % Moisture by KF when stored at 5° C. Time (months)Batch A Batch B Batch C Batch R Batch S 0 0.5 0.6 0.6 0.8 1.0 1 0.5 0.40.6 3 0.5 0.6 0.6 6 0.6 0.7 0.5 0.8 1.3 12 0.6 0.6 0.7 0.9 0.9 24 0.50.7 0.7 0.9 0.9 30 0.7 0.7

TABLE 12 Change in Percent Monomer by SEC when stored at 25° C./60% RHTime (months) Batch A Batch B Batch C Batch R Batch S 0 99.8 99.8 99.898.9 98.8 1 99.8 99.1 99.2 98.7 98.7 3 99.8 99.0 99.0 98.6 98.5 6 99.899.7 99.7 98.9 98.9 9 99.0 99.1 99.1 99.1 99.1 12 99.3 98.9 98.9 98.898.9 15 99.3 99.0 99.0 18 99.4 99.3 99.3 98.7 98.9 24 99.2 99.1 99.198.9 98.9 30 99.0 99.0

TABLE 13 Change in Percent Aggregates by SEC when stored at 25° C./60%RH Batch Batch Batch Batch Batch Time (months) A B C R S 0 0.1 0.1 0.10.2 0.2 1 0.2 0.2 0.2 0.2 0.2 3 0.2 0.3 0.2 0.3 0.3 6 0.2 0.3 0.3 0.20.2 9 0.2 0.3 0.3 0.2 0.2 12 0.2 0.2 0.2 0.3 0.3 15 0.3 0.3 0.3 18 0.30.3 0.3 0.3 0.2 24 0.3 0.3 0.3 0.3 0.2 30 0.4 0.3

TABLE 14 Change in Percent Major Isoform by CEX when stored at 25°C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 68.669.9 69.5 71.7 71.6 1 67.2 68.4 68.6 71.2 71.0 3 68.1 68.6 68.2 70.370.3 6 65.9 67.8 67.8 71.5 71.1 9 69.3 67.5 66.3 68.6 69.0 12 66.7 67.567.4 70.1 70.2 15 66.2 66.6 66.8 18 66.1 65.8 64.9 70.0 70.3 24 66.768.4 68.2 70.6 70.1 30 67.2 67.2

TABLE 15 Change in Percent Acidic Isoforms by CEX when stored at 25°C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 22.820.8 21.4 20.3 20.6 1 21.9 21.8 22.2 21.4 21.6 3 21.7 22.2 22.8 21.822.0 6 22.6 22.9 23.5 21.1 21.4 9 19.9 22.1 23.1 21.8 21.8 12 23.0 21.422.0 21.3 21.3 15 22.5 22.1 22.7 18 22.6 22.1 22.6 21.3 21.5 24 21.721.9 22.6 20.7 20.7 30 22.7 23.2

TABLE 16 Change in Percent Basic Isoforms by CEX when stored at 25°C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 8.59.3 9.1 8.1 7.8 1 10.8 9.8 9.2 7.4 7.3 3 10.3 9.3 9.0 7.8 7.7 6 11.5 9.38.7 7.4 7.5 9 10.8 10.4 10.6 9.7 9.3 12 10.3 11.1 10.7 8.7 8.5 15 11.311.2 10.6 18 11.2 12.1 12.5 8.7 8.2 24 11.6 9.7 9.1 8.7 9.2 30 10.2 9.6

TABLE 17 Change in % (H + L) by Reduced-SDS Page when stored at 25°C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 98 9898 96 96 1 98 98 98 98 98 3 97 98 98 98 98 6 97 97 97 97 97 9 97 97 9798 98 12 98 96 96 98 98 15 97 97 97 18 98 97 97 99 99 24 98 97 98 99 9930 97 98

TABLE 18 Change in Binding Efficacy when stored at 25° C./60% RH Time(months) Batch A Batch B Batch C Batch R Batch S 0 107 106 105 93 102 1115 103 109 3 92 113 100 96 94 6 109 89 97 101 114 9 97 89 85 97 102 1283 91 123 15 96 91 96 18 106 123 87 92 102 24 103 82 90 98 94 30 84 114

TABLE 19 Change in % Moisture by KF when stored at 25° C./60% RH Time(months) Batch A Batch B Batch C Batch R Batch S 0 0.5 0.6 0.6 0.8 1.0 10.5 0.6 0.5 3 0.5 0.7 0.6 6 0.5 0.7 0.7 1.3 1.2 12 0.6 0.8 0.6 0.9 1.024 0.7 0.8 0.6 1.1 1.0 30 0.8 0.7

Cation Exchange Chromatography (CEX)

A phosphate/sodium chloride gradient on a weak cation exchange column isused in a high performance liquid chromatography system to separatecharged species in anti-α4β7 antibody formulations and determine thecharge composition of the antibody species. Acidic Isoforms elute beforethe Major Isoform and Basic Isoforms elute after the Major Isoform.

Stability data for all vedolizumab batches generated using a CEX assayis presented in Tables 3, 6-8 and 14-16. The Tables show, that at thesestorage conditions, there was no trend of reducing the % Major Isoformbelow 55.0%.

Size Exclusion Chromatography (SEC)

SEC is performed using an analytical SEC column (Tosoh Bioscience, LLC,King of Prussia, Pa.). The mobile phase is a phosphate-buffered salinesolution and the absorbance is monitored at 280 nm.

Stability data generated using an SEC assay is presented in Tables 1, 2,4, 5, 12 and 13. The Tables show that none of the listed storageconditions resulted in lowering the % Monomer below 96.0%. Similarly,the % Aggregates remained ≦2.5% for all batches at all listed storageconditions.

SDS-PAGE Assay

SDS-PAGE is performed using an Invitrogen (Carlsbad, Calif.)Tris-Glycine gel, 4-20% for reducing condition and 4-12% fornon-reducing condition. The reconstituted antibody formulation sample isdiluted in liquid formulation buffer then diluted one to two withTris-Glycine SDS Sample Buffer (2×, Invitrogen) either with 10%2-mercaptoethanol (reducing sample buffer) or without 2-mercaptoethanol(non-reducing sample buffer). Samples are briefly heated and loaded incomparison with a molecular weight marker (Invitrogen). The gels arestained with colloidal coomassie blue (Invitrogen) according to themanufacturer's instruction. Protein bands are analyzed by densitometryto identify the % heavy and light chain for reduced gels and % IgG fornon-reduced gels.

Stability data generated using a Reduced SDS-PAGE assay are presented inTables 9 and 17. No noticeable changes were observed for the %Heavy+Light (H+L) chains at all storage conditions listed for allstability lots. The banding pattern was similar to that of the referencestandard and % (H+L) remained at a level ≧90%.

Binding Efficacy

HuT78 cells (human T cell lymphoma cells, American Type CultureCollection, Manassas, Va.) suspended in 1% BSA in PBS, 0.01% sodiumazide are contacted with serial dilutions of primary test antibody.After incubation on ice, the cells are washed and treated withfluorescently labeled secondary antibody. After a further wash, thecells are fixed and suspended in FACS reagent for analysis by flowcytometry (Becton Dickinson Franklin Lakes, N.J.); also see U.S. Pat.No. 7,147,851.

Binding efficacy of vedolizumab was measured relative to the ReferenceStandard and reported as % Reference Standard and EC50. Stability datais presented in Tables 10 and 18. Data for the % Reference Standardshowed variability but remained within the specification limits at allstorage conditions. No evaluated lots of vedolizumab displayed a trendof diminished binding efficacy at the storage conditions listed.

Moisture by Karl Fischer

The formulation is titrated with methanol for a coulometric Karl Fischermoisture determination. Moisture data is presented in Tables 11 and 19.All evaluated lots of vedolizumab had less than 5% moisture at alllisted storage conditions.

Capillary Isoelectric Focusing (cIEF)

cIEF is performed using an iCE280 whole column detection cIEF system(Convergent Biosciences, Toronto, Ontario). Choice of ampholyte can beas recommended by the manufacturer or can be a combination ofcommercially available ampholytes. A useful combination is a mixture of3-10 and 5-8 PHARMALYTE™ (GE Healthcare, Piscataway, N.J.).

Example 3: Modeling the Scale-Up of the Lyophilization Process

Quality by design was used while manipulating the load in the freezedryer and the solids content of the formulation. The load was variedfrom 33 to 100%. The formulation solids content was varied from 9 to 27%by including in the loads a formulation which was 0.5×, 1.0× and 1.5× ofthe target formulation. These formulations had similar T_(g′). Withhigher % solids, the primary drying time increased. In addition, athigher solids content, the product temperature increased due to largerR_(p). The load also has an effect on both stages of drying (FIG. 8).

Example 4: Non-Clinical Safety Study

A study was designed to compare the effect of natalizumab andvedolizumab on immune surveillance of the CNS in Rhesus EAE. Eightanimals were dosed with a placebo control, once weekly. Seven animalswere dosed at 30 mg/kg, once weekly, with natalizumab. Seven animalswere dosed at 30 mg/kg, once weekly, with vedolizumab. The clinicalsymptoms of EAE were observed; the frequency and ratio of leukocytesubsets in CSF were measured by flow cytometry; the total T2 lesion loadin the brain was measured using MRI; and lesion load and demyelinationof the brain was measured using histopathology.

Vedolizumab did not delay onset of clinical symptoms of EAE as comparedto placebo control. It did not inhibit the incidence of EAE, nor themagnitude of clinical scores. Natalizumab significantly (p<0.05) delayedthe onset of clinical symptoms of EAE as compared to placebo control. Itinhibited the incidence of EAE and the magnitude of clinical scores.(FIG. 9)

Vedolizumab did not prevent infiltration of the CSF by leukocytes, Tlymphocytes (helper T lymphocytes, cytotoxic T lymphocytes), Blymphocytes, natural killer cells, or monocytes. In contrast,natalizumab inhibited infiltration of the CSF

Vedolizumab did not inhibit the accumulation of brain lesions, asdetected by increased T2 and decreased MTR values via MRI. Natalizumabprevented lesion formation in all but one animal. Significant (p<0.05)inhibition in brain infiltrates and demyelination was measured byhistology.

The α4β7 integrin was saturated by vedolizumab during the investigation,as shown by a competitive binding assay between vedolizumab dosed invivo and an analytical anti-α4β7 monoclonal antibody added ex vivo. Theanalytical anti-α4β7 mAb does not bind to memory helper T lymphocytes inanimals dosed with vedolizumab. The lack of effect of vedolizumab in theCNS is therefore due to the gastrointestinal-tropic biology of the α4β7integrin.

In summary, vedolizumab (an α4β7 antagonist) does not inhibit EAR Incontrast, natalizumab (α4β1 and α4β7 antagonist) does inhibit EAE. Theα4β1 integrin mediates infiltration of the CNS in EAE. Thus, vedolizumabmay have a lower risk of predisposing patients to PML than natalizumabbecause it does not antagonize the α4β1 integrin and impair immunesurveillance of the CNS in Rhesus EAE.

Example 5: Phase I Clinical Study with Vedolizumab

Forty-nine healthy subjects were randomized and received a single doseof study medication: 39 subjects received vedolizumab (5 mg/mL antibody,20 mM citrate/citric acid, 125 mM sodium chloride, 0.05% polysorbate 80,pH 6.0 (stored long term −70° C. and up to 3 months at −20° C.)) and 10subjects received placebo. Of the 39 subjects who received vedolizumab,8 subjects each received a dose at 0.2, 2.0, 6.0, and 10.0 mg/kg and 7subjects received vedolizumab at 0.5 mg/kg. All 49 subjects completedthe study.

There were no notable differences across vedolizumab cohorts for anydemographic or baseline characteristic. Mean age ranged from 35.4 to51.0 years; individual subject ages ranged from 21 to 63 years.

PK Results

Vedolizumab was administered as a 30-minute intravenous infusion of 0.2to 10.0 mg/kg. The Cmax and area under the serum drug concentration-timecurve of (AUC) values increased with increasing dose. The dose-correctedCmax was approximately the same across cohorts, indicating doseproportionality for this parameter. The dose-normalized area under theserum drug concentration value from time zero to infinity (AUC_(0-inf))increased with increasing dose up to 2.0 mg/kg, indicating there was anonlinear increase in AUC_(0-inf) with increasing dose over the lowerrange of doses administered in this study. Thereafter, AUC_(0-Inf)increased proportionally with dose, indicating linearity of AUC_(0-inf)over the dose range 2.0 to 10.0 mg/kg. The increase in AUC_(0-inf) wasapproximately 2.4-fold higher than expected at the 10.0 mg/kg dosecompared with the 0.2 mg/kg dose.

Similarly, estimates of clearance, volume of distribution, and terminalhalf-life were dose-dependent over the dose range 0.2 to 2.0 mg/kg. Asdose increased, clearance was reduced, distribution volume increased,and, consequently, the terminal elimination half-life was prolonged.However, from 2 to 10.0 mg/kg, there was no apparent change in theseparameters, which suggests a saturation of a rapid elimination processfor vedolizumab at low concentrations. Slower linear eliminationprocesses likely account for a large fraction of clearance ofvedolizumab at higher doses.

In some subjects who developed HAHA to vedolizumab, a faster clearanceof vedolizumab was observed as compared to the HAHA-negative subjectswithin the respective dose level.

TABLE 20 Overview of Vedolizumab PK by Dose Cohort Following IVAdministration of 0.2-10.0 mg/kg Vedolizumab in Healthy Subjects (PKAnalysis Set) VDZ Geometric % Parameter dose N Mean SD Mean CV MedianMin Max C_(max) 0.2 4 5.65 0.629 5.62 11.1 5.45 5.13 6.56 (μg/mL) mg/kg0.5 4 10.6 2.09 10.4 19.7 10.6 8.07 13.1 mg/kg 2.0 7 59.3 11.6 58.4 19.658.4 47.6 78.4 mg/kg 6.0 6 151 19.1 150 12.6 157 120 168 mg/kg 10.0 7243 22.1 243 9.07 242 213 281 mg/kg AUC_(0-tlast) 0.2 4 31.6 4.98 31.315.8 31.6 25.7 37.5 (day*μg/mL) mg/kg 0.5 4 127 48.0 119 37.9 129 70.9178 mg/kg 2.0 7 964 147 955 15.2 972 772 1170 mg/kg 6.0 6 3090 749 302024.2 2830 2360 4100 mg/kg 10.0 7 4870 624 4840 12.8 4750 4120 5870 mg/kgAUC_(0-inf) 0.2 4 39.5 5.79 39.1 14.7 40.2 31.7 45.7 (day*μg/mL) mg/kg0.5 4 134 48.9 127 36.5 134 79.2 188 mg/kg 2.0 7 979 146 969 14.9 993784 1180 mg/kg 6.0 6 3100 750 3030 24.2 2840 2390 4110 mg/kg 10.0 7 4880637 4850 13.0 4750 4130 5920 mg/kg V_(z)(L) 0.2 4 4.02 0.151 4.02 3.764.03 3.83 4.18 mg/kg 0.5 4 4.92 0.620 4.89 12.6 4.66 4.52 5.84 mg/kg 2.07 3.34 0.665 3.28 19.9 3.23 2.29 4.27 mg/kg 6.0 6 2.98 0.644 2.92 21.62.98 2.06 3.98 mg/kg 10.0 7 2.89 1.02 2.73 35.2 2.98 1.49 4.58 mg/kg CL(L/day) 0.2 4 0.413 0.042 0.412 10.1 0.395 0.388 0.476 mg/kg 0.5 4 0.3100.106 0.297 34.3 0.291 0.212 0.446 mg/kg 2.0 7 0.165 0.018 0.164 10.70.162 0.145 0.194 mg/kg 6.0 6 0.140 0.031 0.136 22.0 0.145 0.083 0.166mg/kg 10.0 7 0.140 0.024 0.139 16.9 0.135 0.103 0.171 mg/kg t_(1/2) (day0.2 4 6.79 0.736 6.76 10.8 6.95 5.79 7.47 mg/kg 0.5 4 11.7 2.83 11.424.2 11.4 9.09 14.8 mg/kg 2.0 7 14.1 2.67 13.9 18.9 14.3 10.6 17.5 mg/kg6.0 6 15.1 3.15 14.8 20.9 14.0 11.9 20.3 mg/kg 10.0 7 14.8 7.38 13.749.8 12.5 8.26 30.7 mg/kg Abbreviations: AUC_(0-inf) = area under thedrug concentration-time curve, extrapolated to infinity; AUC_(0-tlast) =area under the drug concentration-time curve from administration time tothe last measurement time point at which the concentration is above thelower limit of quantification; CL = total clearance; C_(max) = maximumdrug concentration; t_(1/2) = terminal half-life; V_(z) = volume ofdistribution based on the terminal phase.

After reaching C_(max), serum concentrations of Vedolizumab fell in agenerally monoexponential fashion until concentrations reachedapproximately 1 to 10 mg/L. Thereafter, concentrations appeared to fallin a nonlinear fashion.

The C_(max) and AUC values increased with increasing dose. For theavailable data, the dose-corrected Cmax was approximately the sameacross cohorts, indicating dose proportionality for this parameter. Thedose-normalized AUC_(0-inf) increased with increasing dose up to 2.0mg/kg, indicating there was a nonlinear increase in AUC_(0-inf) withincreasing dose over the lower range of doses administered in thisstudy. Thereafter, AUC_(0-inf) increased proportionally with dose,indicating linearity of AUC0-inf over the dose range 2.0 to 10.0 mg/kg.The increase in AUC_(0-inf) was approximately 2.4-fold higher thanexpected at the 10.0 mg/kg dose compared with the 0.2 mg/kg dose.

PD Results

The PD parameters of Vedolizumab following a 30-minute intravenousinfusion of 0.2 to 10.0 mg/kg vedolizumab by cohort are summarized inTable 21 and Table 22 for Act-1 and MAdCAM respectively.

TABLE 21 Overview of Vedolizumab Pharmacodynamics, Percent Inhibition of% Act- 1⁺ [CD4⁺ CD45RO^(high)], by Dose Cohort Following IVAdministration of 0.2-10.0 mg/kg Vedolizumab in Healthy Subjects (PDAnalysis Set) VDZ Geometric Parameter dose N Mean SD Mean % CV MedianMin Max E_(max) 0.2 4 99.6 0.387 99.6 0.388 99.6 99.1 100 (% Inhibition)mg/kg 0.5 4 99.5 0.599 99.5 0.602 99.5 98.9 100 mg/kg 2.0 6 99.9 0.17299.9 0.172 100 99.6 100 mg/kg 6.0 6 100 0.000 100 0.000 100 100 100mg/kg 10.0 6 99.7 0.326 99.7 0.327 99.8 99.3 100 mg/kg AUEC_(0-inf) 0.24 4030 1010 3920 25.2 4090 2760 5160 (% Inhibition*d) mg/kg 0.5 4 64301450 6300 22.6 6530 4860 7810 mg/kg 2.0 6 13200 623 13200 4.72 1290012800 14500 mg/kg 6.0 6 16700 3030 16500 18.1 16300 13300 20100 mg/kg10.0 6 19300 644 19300 3.33 19600 18200 19900 mg/kg AUEC_(0-inf) = areaunder the drug effect versus time curve from time 0 to the time of thelast non-zero concentration; E_(max) = maximum drug effect

TABLE 22 Overview of Vedolizumab Pharmacodynamics, Percent Inhibition of% MADCAM⁺ [CD4⁺ CD45RO^(high)], by Dose Cohort Following IVAdministration of 0.2-10.0 mg/kg Vedolizumab in Healthy Subjects (PDAnalysis Set) VDZ Geometric % Parameter dose N Mean SD Mean CV MedianMin Max E_(max) 0.2 4 99.2 0.537 99.2 0.542 99.4 98.4 99.6 (%Inhibition) mg/kg 0.5 4 99.6 0.323 99.6 0.324 99.5 99.3 100 mg/kg 2.0 699.7 0.365 99.7 0.366 99.7 99.2 100 mg/kg 6.0 6 99.8 0.279 99.8 0.280100 99.4 100 mg/kg 10.0 6 100 0.000 100 0.000 100 100 100 mg/kgAUEC_(0-inf) 0.2 4 4000 576 3970 14.4 4210 3160 4440 (% Inhibition*d)mg/kg 0.5 4 6770 1400 6660 20.6 6840 5170 8230 mg/kg 2.0 6 13000 79613000 6.12 13000 11700 13900 mg/kg 6.0 6 16200 3320 15900 20.5 1580011800 20000 mg/kg 10.0 6 17700 1330 17700 7.5 17700 16500 19000 mg/kgAUEC_(0-inf) = area under the drug effect versus time curve from time 0to the time of the last non-zero concentration; E_(max) = maximum drugeffect

Vedolizumab inhibited the PD parameters, Act-1 and MAdCAM-1-Fc, nearlymaximally at all time points where vedolizumab was measurable in serum.Once vedolizumab concentrations decreased below the limit of detectionof the assay, the inhibition of Act-1 and MAdCAM-1-Fc returned toapproximately the baseline level.

In some subjects who developed HAHA to vedolizumab, a faster loss ofα4β7 receptor saturation was observed as compared to the HAHA-negativesubjects in the respective dose level.

Safety Results

Vedolizumab was generally safe and well tolerated at single IV doses upto 10.0 mg/kg. No deaths, serious adverse events (SAEs) or AEs leadingto study discontinuation occurred during the study.

Immunogenicity/Human Antihuman Antibody (HAHA) Formation

One (10%) subject in the placebo group and 21 (54%) subjects in thecombined vedolizumab dose groups had a positive HAHA at some pointduring the study. Although positive HAHA samples were observed in alldose cohorts, HAHA titers >125 were found only in the 2 lowestvedolizumab dose groups. Dose-dependent suppression of HAHA formationhas been observed previously with vedolizumab. Nineteen of the 22vedolizumab-treated subjects who were HAHA-positive had neutralizingHAHA present.

TABLE 23 Overview of Human Antihuman Antibodies Findings: SafetyPopulation 0.2 mg/kg 0.5 mg/kg 2.0 mg/kg 6.0 mg/kg 10.0 mg/kg CombinedPlacebo VDZ VDZ VDZ VDZ VDZ VDZ N = 10 N = 8 N = 7 N = 8 N = 8 N = 8 N =39 Subjects 10  8 7 8 8 8 39 Tested Any HAHA 1 (10) 6 (75) 4 (57) 2 (25)3 (38) 6 (75) 21 (54) Positive, n (%) Highest 1 (10) 4 (50) 2 (29) 2(25) 3 (38) 6 (75) 17 (44) HAHA Titer < 125, n(%) Highest 0 2 (25) 2(29) 0 0 0  4 (10) HAHA Titer ≧ 125, n(%) Any 0 5 (63) 4 (57) 2 (25) 3(38) 5 (63) 19 (49) Neutralizing HAHA Positive, n(%) Highest 0 3 (38) 2(29) 2 (25) 3 (38) 5 (63) 15 (38) Neutralizing HAHA Titer < 125, n(%)Highest 0 2 (25) 2 (29) 0 0 0  4 (10) Neutralizing HAHA Titer ≧ 125,n(%)

One subject in the placebo group and 11 subjects in the vedolizumabgroup were persistently HAHA-positive.

TABLE 24 Overall Human Antihuman Antibody Status (Safety Population) 0.2mg/kg 0.5 mg/kg 2.0 mg/kg 6.0 mg/kg 10.0 mg/kg Combined Placebo VDZ VDZVDZ VDZ VDZ VDZ N = 10 N = 8 N = 7 N = 8 N = 8 N = 8 N = 39 HAHA 9 (90) 2 (25) 3 (43) 6 (75) 5 (63) 2 (25) 18 (46) negative^(a) n(%) Isolated 02 (25) 1 (14) 1 (13) 1 (13) 5 (63) 10 (26) HAHA^(b) n(%) Persistent 1(10)_(—) 4 (50) 3 (43) 1 (13) 2 (25) 1 (13) 11 (28) HAHA^(c) n(%)^(a)HAHA Negative: Subjects with no positive HAHA results ^(b)IsolatedHAHA: Subjects with only 1 positive HAHA sample with titer < 25^(c)Persistent HAHA: Subjects with 2 or more positive HAHA samples, or 1positive sample with titer ≧ 25

Conclusions

This phase 1 study characterized the PK/PD and initial safety profilesof vedolizumab derived from CHO cells. The results of this study wereused to support dose selection for phase 3 pivotal trials ininflammatory bowel disease.

Vedolizumab demonstrated dose proportionality over the tested dose rangefor the Cmax parameter; however, dose-dependent changes in AUC0-inf, CL,Vz, and t½ were observed from 0.2 to 2.0 mg/kg, suggesting nonlinear PKbehavior of vedolizumab. At dose levels greater than 2.0 mg/kg, nofurther changes in these parameters were observed, which suggests asaturation of a rapid elimination process for vedolizumab at lowconcentrations. Slower linear elimination processes likely account for alarge fraction of clearance of vedolizumab at higher doses.

Vedolizumab inhibited the PD parameters, Act-1 and MAdCAM-1-Fc, at ornear maximal levels at all time points when vedolizumab was measurablein serum. Once vedolizumab concentrations decreased below the limit ofdetection of the assay, the inhibition of Act-1 and MAdCAM-1-Fc returnedto approximately the baseline level.

In some subjects who developed HAHA to vedolizumab, a faster clearanceof vedolizumab and loss of α4β7 receptor saturation was observed ascompared to the HAHA-negative subjects within the respective dose level.

Vedolizumab was well-tolerated. No deaths, SAEs, or AEs leading todiscontinuation of study drug administration occurred during the study,nor were any dose-toxicity relationships observed. No systemicopportunistic infections (including PML) or neoplasms were reported.

Unlike nonspecific α4 antagonists, vedolizumab was not associated withlymphocytosis or mean increases in circulating eosinophils, basophils,or monocytes, nor was there any evidence of depletion of lymphocytes.

Vedolizumab did elicit the formation of HAHA, but the highest titers(>125) were observed only in the 2 lowest dose groups, a finding thatsupports previous observations of a dose-dependent reduction inimmunogenicity. These data show that the administration of higher dosesof vedolizumab may minimize clinically significant HAHA formation.

In conclusion, vedolizumab was generally safe and well tolerated whenadministered in single doses of 0.2 to 10.0 mg/kg to healthy subjects.

Example 6: Determination of the Effect of Vedolizumab on the CD4:CD8Ratio

Healthy subjects ages 18-45 were treated with a single 450 mg dose ofvedolizumab reconstituted from a lyophilized formulation of 10% sucroseand diluted into an infusion system of 0.9% saline. Cerebrospinal fluid(CSF) was collected by lumbar puncture before (baseline) and 5 weeksafter the single 450-mg dose of vedolizumab. Each subject served ashis/her own control.

A 5-week time point was selected based on a previous study that showedpatients with MS treated with natalizumab demonstrated effects on CSFCD4+:CD8+ lymphocyte ratio and reduction in number of brain lesionsafter only one dose (Stuve et al. Arch Neurol. 2006; 63:1383-1387; Stuveet al. Ann Neurol. 2006; 59:743-747. Miller et al. N Engl J. Med. 2003;348(1):15-23); and also because at 5 weeks, a 450-mg dose of vedolizumabis sufficient to saturate the target and provides serum concentrationsthat exceed estimated steady-state trough levels associated with thephase 3 dose regimen of 300 mg every 4 weeks.

Approximately 15 mL CSF was obtained from each subject forimmunophenotyping. CSF samples were included for analyses if they metthe following criteria: ≦10 RBCs/μL per sample (to minimize peripheralblood contamination); negative CSF culture result; adequate T-lymphocytenumbers in each flow cytometry sample; and no detection of serumantibodies to vedolizumab.

Week 5 median (34.80 μg/mL) and individual subject serum vedolizumabconcentrations (range 24.9-47.9 μg/mL) were higher than projectedsteady-state trough concentration (˜24 μg/mL) for the phase 3 doseregimen. A high degree (>90%) of α4β7 receptor saturation was observedat week 5 as measured by MAdCAM-1-Fc, indicating vedolizumab'ssaturation of its target at the time of endpoint assessment.

Vedolizumab was not detected in any CSF sample (detection limit=0.125μg/mL).

Effect on CD4+ and CD8+T Lymphocyte Numbers and Ratio

Vedolizumab did not significantly reduce CD4+:CD8+ ratio (Table 25).None of the subjects had a postdose CD4+:CD8+ ratio <1 (p<0.0001(1-sided t-test)). Vedolizumab did not significantly reduce the numberof CD4+ or CD8+T lymphocytes in CSF. In addition, there were nosignificant changes in CSF % CD4+ and % CD8+T lymphocytes (Table 26).Also, no significant changes in peripheral blood WBC, CD4+ and CD8+memory T lymphocytes (Table 27) were observed.

TABLE 25 Effect of Treatment on CSF CD4+:CD8+ Ratio (EvaluablePopulation, n = 13) CD4+:CD8+ Ratio Baseline Week 5 Difference†CD4+:CD8+ ratio 3.59 (0.273) 3.60 (0.265)* 0.01 (0.197) Mean (SE) Range1.53-5.67 1.42-5.15 90% 2-sided CI for 3.00-4.19 3.132, 4.077 ratio 90%2-sided CI for −0.337, 0.363 difference CI = confidence interval *p <0.0001 (one sided one sample t-test for H0: μ < 1 vs H1: μ >= 1).†Difference is defined as week 5 ratio minus baseline ratio

TABLE 26 Treatment Effect on CSF CD4+ and CD8+ Lymphocyte Count(Evaluable Population, n = 13) Baseline Week 5 CD4+ as % of 75.160(7.3831) 74.215 (6.3732) Lymphocytes, mean (SD) CD8+ as % of 22.272(5.4320) 22.007 (6.1624) Lymphocytes, mean (SD)

TABLE 27 Peripheral Blood Memory T Lymphocytes (RO+) Counts (EvaluablePopulation, n = 13) Baseline Week 5 Mean (SD) Mean (SD) CD4 + CD45RO+27.85 (4.98) 27.06 (5.02) CD8 + CD45RO+(%) 11.24 (3.40) 10.78 (2.98)

Summary

Vedolizumab did not affect CSF CD4+ and CD8+ cell counts or CD4+:CD8+ratio in healthy volunteers after a single 450 mg dose. None of thesubjects had a reduction in the post-dose CSF CD4+:CD8+ ratio to lessthan 1. Vedolizumab was not detected in CSF. In addition, there was nochange observed in the total WBCs or memory T lymphocyte CD4+ and CD8+subsets in peripheral blood. Saturation of the target (α4β7) in bloodoccurred in all subjects at the time of endpoint assessment. The CSFCD4+ and CD8+ lymphocyte levels and ratio were similar to thosepreviously reported in the literature.

These results are consistent with vedolizumab's lack of effect on bothphysiologic CNS immune surveillance and pathologic CNS inflammation ofmonkeys (See Example 4).

Example 7: Long-Term Clinical Experience with Vedolizumab for theTreatment of IBD

A phase 2 open-label safety extension study was completed to assess thelong-term pharmacokinetics (PK), pharmacodynamics (PD), safety, andefficacy of vedolizumab. Patients were aged 18 to 75 years old, and hadeither previously participated in an earlier PK/PD/safety study inulcerative colitis patients or had IBD symptoms for at least 2 monthsconfirmed endoscopically and/or histopathologically and/orradiologically within 36 months of screening.

All patients received an intravenous dosing regimen of either 2 mg/kg or6 mg/kg of vedolizumab (5 mg/mL antibody, 20 mM citrate/citric acid, 125mM sodium chloride, 0.05% polysorbate 80, pH 6.0 (stored long term −70°C. and up to 3 mo −20° C.)) on days 1, 15 and 43, followed by a doseevery 8 weeks for up to a total of 78 weeks. Patients were eithertreatment-naïve ulcerative colitis or Crohn's disease patients, orulcerative colitis patients that had participated in an earlier clinicaltrial.

Efficacy/quality of life (QoL); partial Mayo score (PMS), Crohn'sdisease activity index (CDAI), and Inflammatory Bowel DiseaseQuestionnaire (IBDQ) were used to assess the results of the study.

PK Results

Mean pre-infusion vedolizumab concentrations were dose proportional, andremained steady and detectable throughout the study.

PD Results

Receptors (% ACT-1+[CD4+CD45RO HIGH] and % MADCAM+[CD4+CD45RO HIGH] werealmost fully inhibited throughout the study period at all dose levels.

Partial Mayo Score

Baseline mean PMS was higher for treatment-naïve ulcerative colitispatients (5.4) than for ulcerative colitis rollover patients (2.3). Byday 43, mean PMS showed a pronounced decrease for both rollover andtreatment-naïve ulcerative colitis patients. By day 155, mean scores ofthe two groups were similar. Mean PMS continued to decrease through day267, and leveled off thereafter.

Crohn's Disease Activity Index

CD patients' mean CDAI decreased from 294.6 at baseline to 237.7 at Day43, and continued to decrease through day 155 (156.1).

IBDQ

Ulcerative colitis rollover patients had the highest mean IBDQ scores atbaseline. By day 43, mean IBDQ scores had increased in all three diseasegroups. Mean IBDQ scores continued to increase over time in all 3disease groups, reaching a maximum at day 155 for Crohn's Diseasepatients, and at day 491 for treatment-naïve ulcerative colitis patientsand ulcerative colitis rollover patients.

C—Reactive Protein

Both ulcerative colitis rollover and Crohn's disease patients showeddecreased mean CRP levels through day 155 and then leveled off.Treatment-naïve ulcerative colitis patients had a lower mean CRP levelat baseline than ulcerative colitis rollover patients (2.28 v. 7.09).Mean CRP levels of the treatment-naïve ulcerative colitis patientsremained relatively constant at all time points assessed.

Other Safety Results

No systematic opportunistic infections (including PML) were reportedduring the study. One patient tested positive for JC viremia at a singletime point, though was negative for JCV at all other time points. Threeof 72 patients (4%) had positive HAHA results (two of these weretransiently positive). The study showed no evidence of liver toxicity,lymphocytosis, or lymphopenia, or any other drug-associated laboratorychanges.

Conclusions

Vedolizumab administered at 2.0 or 6.0 mg/kg once every 8 weeks for upto 78 weeks achieved target receptor saturations, was associated withdurable mean decreases in disease activity and improved IBDQ scores, wasgenerally safe and well tolerated, and demonstrated acceptableimmunogenicity.

Example 8: Induction of Response and Remission in Patients with Moderateto Severely Active Crohn's Disease

A randomized, double blind, placebo controlled multi-center study wascompleted to evaluate the induction effect of vedolizumab at 300 mgdoses (reconstituted from a formulation of 60 mg/ml antibody in 50 mMhistidine, 125 mM arginine, 0.06% polysorbate 80, 10% sucrose, at pH6.3which was lyophilized), in TNFα antagonist failure patients at week 6(after 2 doses—0 and 2 weeks) and at week 10 (after 3 doses). The studyconsisted of 416 patients, 75% of whom were TNFα antagonist failures,and 25% of whom were TNFα naïve. Demographics and concomitant IBDmedication were balanced across treatment groups. Baseline diseasecharacteristics were also balanced across treatment groups, except forbaseline disease activity.

The primary endpoint designated for the study was week 6 remission (%)in anti-TNF-α antagonist failure population. The key secondary endpointsthat were evaluated (sequential testing procedure) were: week 6remission (%) in overall population, week 10 remission (%) in anti-TNF-αantagonist failure and overall population (using Hochberg procedure),week 6 and 10 sustained remission (%) in anti-TNF-α antagonist failureand overall population (using Hochberg procedure), and week 6 enhancedresponse (%) in anti-TNF-α antagonist failure population.

TABLE 28 Baseline CDAI: Placebo Vedolizumab p-value TNF ITT: Mean 306.1(55.43) 316.1 (52.63) 0.0945 (Std Dev) Overall ITT: Mean 301.3 (54.97)313.9 (53.17) 0.0153 (Std Dev)

TABLE 29 Induction Study Results: Primary and Key Secondary EndpointsTNF ITT (N = 315) Overall ITT (N = 416) PLA VDZ Diff PLA VDZ DiffEndpoints N = 157 V = 158 (RR) P-value N = 207 N = 209 (RR) P-valuePrimary 12.1% 15.2%  3.0% (1.2) 0.4332 Wk 6 Remission 1st 12.1% 19.1% 6.9% (1.6) 0.0478 Secondary Wk 6 Remission 2nd 12.1% 26.6% 14.4% (2.2)0.0012  13% 28.7% 15.5% (2.2) <0.0001 Secondary Wk 10 RemissionSustained 8.3% 12.0%  3.7% (1.4) 0.2755  8.2% 15.3%   7% (1.9) 0.0249Remission (both Wk 6&10) Enhanced 22.3% 39.2% 16.9% (1.8) 0.0011Response (CDAI100)

TABLE 30 Results in Anti-TNF-α Antagonist Naïve Patients (n = 101, 24%of overall) Placebo % Vedolizumab % Difference % 95% Cl Remission Week 612 31.4 19.1 (3.3, 35.0) Remission Week 10 16 35.3 19.2 (2.4, 35.8)

TABLE 31 Study Results: Clinical Remission at Weeks 6 and 10, KeySubgroup—Previous Tx Failures, ITT Overall Subgroup Variable Placebo VDZDiff 95% Cl Any prior anti- N 156 155 TNF failure (75% Wk6 Rem 12.8 14.82 (−5.7, of ITT) (%) 9.7) Wk10 Rem 12.8 26.5 13.6 (4.9, (%) 22.3) PriorN 45 44 immunomodulator Wk 6 Rem 11.1 31.8 20.7 (−0.5, failure but not(%) 39.7) anti-TNF failure Wk10 Rem 15.6 31.8 16.3 (−1.1, (21% ITT) (%)33.6) Prior N 5 9 corticosteroid Wk6 Rem 0 33.3 33.3 (−23.9, failureonly (3% (%) 75.7) ITT) Wk10 Rem 0 44.4 44.4 (−13.4, (%) 85.3)

The study showed that TNF-α antagonist failure patients required 3 dosesfor induction of remission. Remission rates in TNF-α antagonist failurepatients increased between week 6 and week 10, but only for thevedolizumab group (not placebo). Remission rates for TNF-α antagonistnaïve patients did not increase substantially between week 6 and 10. Ofthe TNF-α antagonist failure population with a high degree of diseaseseverity, 43% never responded to a TNF-α antagonist, and 45% lostresponse.

Example 9: Induction and Maintenance of Response and Remission inPatients with Moderately to Severely Active Ulcerative Colitis

A single trial comprising two randomized, double blind, multi-centerstudies designed to evaluate induction and maintenance of response andremission in patients with moderately to severely active ulcerativecolitis. Demographic and baseline disease characteristics werecomparable across all treatment groups.

The induction study, using intravenous administration, compared placeboagainst vedolizumab, at a 300 mg dose reconstituted from a lyophilizedformulation of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine,0.06% polysorbate 80, 10% sucrose, at pH 6.3, with an endpoint at 6weeks after 2 doses of vedolizumab.

The maintenance study, using the same formulation and route ofadministration as the induction study, compared placebo againstvedolizumab dosed every four weeks, and placebo against vedolizumabdosed every eight weeks. Each patient was age 18-80, diagnosed withmoderately to severely active ulcerative colitis; demonstrated, over theprevious 5 year period, an inadequate response to, loss of response to,or intolerance of at least one conventional therapy (e.g.corticosteroids); and may be receiving a therapeutic dose ofconventional therapies for IBD. The endpoint of this study was at 52weeks, analyzing the induction responder population. Both phases of thetrial met their primary endpoints, namely, clinical response ininduction and clinical remission in maintenance.

Blood samples were collected to measure concentrations of vedolizumabduring the study. The mean serum concentration of vedolizumab at the endof the induction phase was 20 to 30 μg/mL. The mean vedolizimab troughserum concentrations at steady state after 30 min IV infusion of 300 mgdose administration were between 9 to 13 μg/mL for the q8wks regimen andbetween 35 to 40 μg/mL for the q4wks regimen. At the end of infusion,the vedolizimab median plasma concentrations were between 98 and 101μg/mL for the q8ks (8 weeks) regimen and around 129 and 137 μg/mL forthe q4 wks (4 weeks).

Summaries of the responses of the induction and maintenance studies areprovided in Tables 32-35. A significantly greater proportion ofvedolizumab-treated patients achieved clinical response, remission, andmucosal healing at 6 weeks, compared with placebo (Table 32). 39% of theinduction phase intent-to-treat population had prior anti-TNFα failure.Clinical response and remission rates were higher in vedolizumab thanplacebo patients among both those with prior anti-TNF failure and thosewith no prior anti-TNF exposure. In preliminary analyses through week 6,rates of adverse events (AEs), serious AEs, and adverse events leadingto study discontinuation were higher in the placebo group thanvedolizumab group. A significantly greater proportion of vedolizumabpatients than placebo patients achieved clinical remission, mucosalhealing, and corticosteroid-free remission at 52 wks and durableresponse and remission (Table 33). 32% of the maintenance studypopulation had prior anti-TNFα failure. Clinical remission and durableclinical response rates were greater with vedolizumab than placebo inboth TNF failure and TNF naïve patients. In the safety population(N=895) for wks 0-52, rates of adverse events (AEs), serious AEs, andserious infections were similar between vedolizumab and placebo groups.No increase in rates of opportunistic or enteric infections was observedin the vedolizumab group.

TABLE 32 Induction Study Results—Primary and Key Secondary EndpointsEfficacy Endpoints Placebo Vedolizumab Difference/RR P value Clinical25.5% 47.1% 21.7%/1.8 <0.0001 Response (%) Clinical  5.4% 16.9%11.5%/3.1 0.0010 Remission (%) Mucosal 24.8% 40.9  16.1%/1.6 0.0013Healing (%)

TABLE 33 Maintenance Study Results—Primary and Key Secondary EndpointsEfficacy Endpoint Difference/RR Placebo VDZ Q8 VDZ Q4 Q8 vs. Pb N = 126N = 122 N = 125 Q4 vs. Pb P value Clinical Remission 15.9 41.8 44.826.1/2.7 <0.0001 (%) 29.1/2.8 <0.0001 Durable Response 23.8 56.6 52.0 32.8/2.44 <0.0001 (%) 28.5/2.2 <0.0001 Mucosal Healing 19.8 51.6 56.032.0/2.6 <0.0001 (%) 36.3/2.8 <0.0001 Durable Remission 8.7 20.5 24.011.8/2.4 0.0090 (%) 15.3/2.8 0.0011 Corticosteroid-free 13.9 31.4 45.217.6/2.3 0.0133 Remission (%) n = 72 n = 70 N = 273 31.4/3.3 <0.0001

TABLE 34 Induction Study: Clinical Response and Remission at 6 Weeks inPatients with Prior Anti-TNF-α Antagonist Failure and Without Anti-TNFExposure, ITT Population Patients with Prior Anti-TNF-α AntagonistFailure (39%) Endpoint Placebo Vedolizumab Difference 95% Cl N = 63 N =82 Clinical 20.6 39.0 18.4 3.9, 32.9 Response (%) Clinical 3.2 9.8 6.6−9.8, 22.8  Remission (%) Patients Without Anti-TNF-α AntagonistExposure (55%) Placebo Vedolizumab Difference 95% Cl N = 76 N = 130Clinical 26.3 53.1 26.8 13.7, 39.9 Response (%) Clinical 6.6 23.1 16.5 2.4, 30.2 Remission (%)

TABLE 35 Clinical Remission and Durable Clinical Response at 52 Weeks:Patients with Prior Anti-TNF-α Antagonist Failure or Without Anti-TNF-αAntagonist Exposure ITT Population Patients with Prior Anti-TNF-αAntagonist Failure (32%) Endpoint Placebo VDZ VDZ Difference 95% Cl N =38 Q8Wks Q4Wks Q8wks vs N = 43 N = 40 Placebo Q4 wks vs. PlaceboClinical remission 5.3 37.2 35.0 31.9 10.3, (%) 29.7 51.4 7.4, 49.4Durable Clinical 15.8 46.5 42.5 30.7 11.8, Response (%) 26.7 49.6 7.5,45.9 Patients without Anti-TNF-α Antagonist Exposure (60%) Placebo VDZVDZ Difference 95% Cl N = 79 Q8wks Q4wks Q8wks vs. N = 72 N = 73 PlaceboQ4wks vs. Placebo Clinical Remission 19.0 45.8 47.9 26.8 12.4, (%) 29.041.2 14.6, 43.3 Durable Clinical 26.6 65.3 56.2 38.7 24.0, Response (%)29.6 53.4 14.6, 44.6

Example 10: Induction and Maintenance of Response and Remission inPatients with Moderately to Severely Active Crohn's Disease

A single trial comprising two randomized, double blind, multi-centerstudies designed to evaluate induction and maintenance of response andremission in patients with moderately to severely active Crohn'sDisease. Demographic and baseline disease characteristics werecomparable across all treatment groups.

The induction study, using intravenous administration, compared placeboagainst vedolizumab, at a 300 mg dose reconstituted from a lyophilizedformulation of 60 mg/ml antibody in 50 mM histidine, 125 mM arginine,0.06% polysorbate 80, 10% sucrose, at pH 6.3, with an endpoint at 6weeks after 2 doses of vedolizumab.

The maintenance study, using the same formulation and route ofadministration as the induction study, compared placebo againstvedolizumab dosed every four weeks, and placebo against vedolizumabdosed every eight weeks. The endpoint of this study was at 52 weeks,analyzing the induction responder population.

Surprisingly, this study showed that Q4 and Q8 week groups yielded verysimilar results. Summaries of the responses of the induction andmaintenance studies are provided in Tables 36-39. A significantlygreater proportion of vedolizumab-treated patients achieved clinicalremission and enhanced response, compared with placebo (Table 36).Clinical remission and enhanced response rates were higher invedolizumab than placebo patients among both those with prior anti-TNFfailure and those with no prior anti-TNF exposure. Rates of adverseevents (AEs), serious AEs, and serious infections were similar betweenvedolizumab and placebo groups. No increase in rates of opportunistic orenteric infections was observed in the vedolizumab group.

TABLE 36 Induction Study Results—Primary and Secondary EndpointsEndpoints Placebo Vedolizumab Adjusted N = 148 N = 220 Difference/RR Pvalue Clinical  6.8% 14.5% 7.8%/2.1 0.0206 Remission (%) Enhanced 25.7%31.4% 5.7%/1.2 0.2322 Response (%) Mean CRP −3.6 −2.9 0.9288 Change N =147 N = 220 (μg/mL)

TABLE 37 Maintenance Study Results—Primary and Key Secondary EndpointsEfficacy Endpoint Adj. Difference/RR Placebo VDZ Q8 VDZ Q4 Q8 vs. Pb P N= 153 N = 154 N = 154 Q4 vs. Pb value Clinical Remission 21.6 39.0 36.417.4/1.8 0.0007 (%) 14.7/1.7 0.0042 Enhanced Response 30.1 43.5 45.513.4/1.4 0.0132 (%) 15.3/1.5 0.0053 Corticosteroid-free 15.9 31.7 28.815.9/2.0 0.0154 Remission (%) N = 82 N = 82 N = 80 12.9/1.8 0.0450Durable Remission 14.4 21.4 16.2  7.2/1.5 0.1036 (%)  2.0/1.1 0.6413

TABLE 38 Clinical Remission and Enhanced Response at 6 Weeks in Patientswith Prior Anti-TNF-α Antagonist Failure and Without Anti-TNF Exposure,ITT Population Patients with Prior Anti-TNF-α Antagonist Failure (48%)Endpoint Placebo Vedolizumab Difference 95% Cl N = 70 N = 105 Clinical4.3 10.5 6.2 (−9.1, 21.3) Remission (%) Enhanced 22.9 23.8 1.0 (−11.8,13.7)  Response (%) Patients Without Anti-TNF-α Antagonist Exposure(50%) Placebo Vedolizumab Difference 95% Cl N = 76 N = 130109 Clinical9.2 17.4 8.2 (−1.4, 17.9) Remission (%) Enhanced 30.3 42.2 11.9 (−1.9,25.8) Response (%)

TABLE 39 Clinical Remission and Enhanced Response at 52 Weeks: Patientswith Prior Anti-TNF-α Antagonist Failure or Without Anti-TNF-αAntagonist Exposure ITT Population Patients with Prior Anti-TNF-αAntagonist Failure (51%) Endpoint Placebo VDZ VDZ Difference 95% Cl N =78 Q8Wks Q4Wks Q8wks vs N = 82 N = 77 Placebo Q4 wks vs. PlaceboClinical remission 12.8 28.0 27.3 15.2 (3.0, (%) 14.5 27.5) (2.0, 26.9)Enhanced 20.5 29.3 37.7 8.8 (−4.6, Response (%) 17.1 22.1) (3.1, 31.2)Patients without Anti-TNF-α Antagonist Exposure (45%) Placebo VDZ VDZDifference 95% Cl N = 71 Q8wks Q4wks Q8wks vs. N = 66 N = 71 PlaceboQ4wks vs. Placebo Clinical 26.8 51.1 46.5 24.8 (8.9, Remission (%) 19.740.6) (4.2, 35.2) Enhanced 38.0 60.6 53.5 22.6 (6.3, Response (%) 15.538.9) (−0.7, 31.7)

TABLE 40 Summary of Sequences SEQ ID NO: Sequence Shown Description  1FIG. 1 DNA encoding heavy chain of humanized anti- α4β7 immunoglobulin 2 FIG. 1 Amino acid sequence of heavy chain of humanized anti-α4β7immunoglobulin  3 FIG. 2 DNA encoding the light chain of humanized anti-α4β7 immunoglobulin  4 FIG. 2 Amino acid sequence of light chain ofhumanized anti-α4β7 immunoglobulin  5 FIG. 3 Mature humanized lightchain of LDP-02  6 FIG. 4 Generic human kappa light chain constantregion  7 FIG. 4 Generic murine kappa light chain constant region  8Referenced on page 37 CDR1 of heavy chain SYWMH mouse ACT-1 antibody(SEQ ID NO: 8)  9 Referenced on page 38 CDR2 of heavy chainEIDPSESNTNYNQKFKG mouse ACT-1 antibody (SEQ ID NO: 9) 10 Referenced onpage 38 CDR3 of heavy chain GGYDGWDYAIDY mouse ACT-1 antibody (SEQ IDNO: 10) 11 Referenced on page 38 CDR1 of light chain RSSQSLAKSYGNTYLSmouse ACT-1 antibody (SEQ ID NO: 11) 12 Referenced on page 38 CDR2 oflight chain GISNRFS mouse ACT-1 antibody (SEQ ID NO: 12) 13 Referencedon page 38 CDR3 of light chain LQGTHQPYT mouse ACT-1 antibody (SEQ IDNO: 13) 14 FIG. 7 human GM607 CL antibody kappa light chain variableregion 15 FIG. 7 Human 21/28 CL antibody heavy chain variable region

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A stable pharmaceutical formulation comprisingsucrose, at least about 120 mg of an anti-α4β7 antibody, and arginine,wherein the formulation is a lyophilized formulation, wherein theformulation comprises at least 50% major isoform of the antibody asdetermined by Cation Exchange Chromatography (CEX), wherein the molarratio of sucrose to anti-α4β7 antibody (mole:mole) is at least about650:1, wherein the arginine to antibody-molar ratio (mole:mole) isgreater than 250:1, and wherein the antibody is humanized, comprises aheavy chain variable region comprising a complementarity determiningregion 1 (CDR1) as set forth in SEQ ID NO: 8, a CDR2 as set forth in SEQID NO: 9, and a CDR3 as set forth in SEQ ID NO: 10, and comprises alight chain variable region comprising a CDR1 as set forth in SEQ ID NO:11, a CDR2 as set forth in SEQ ID NO: 12, and a CDR3 as set forth in SEQID NO:
 13. 2. The formulation of claim 1, wherein said formulationfurther comprises a buffering agent.
 3. The formulation of claim 2,wherein said buffering agent is histidine.
 4. The formulation of claim1, wherein said formulation further comprises a surfactant.
 5. Theformulation of claim 1, wherein said formulation comprises at leastabout 5% to about 10% anti-α4β7 antibody before lyophilization.
 6. Astable pharmaceutical formulation comprising sucrose, at least about 120mg of an anti-α4β7 antibody, histidine, arginine, and polysorbate 80,wherein the formulation is a lyophilized formulation, wherein theformulation comprises at least 50% major isoform of the antibody asdetermined by Cation Exchange Chromatography (CEX), wherein the molarratio of sucrose to anti-α4β7 antibody (mole:mole) is at least about650:1, wherein the molar ratio of histidine and arginine to antibody(mole:mole) is about 200:1 to about 500:1 and wherein the antibody ishumanized, comprises a heavy chain variable region comprising acomplementarity determining region 1 (CDR1) as set forth in SEQ ID NO:8, a CDR2 as set forth in SEQ ID NO: 9, and a CDR3 as set forth in SEQID NO: 10, and comprises a light chain variable region comprising a CDR1as set forth in SEQ ID NO: 11, a CDR2 as set forth in SEQ ID NO: 12, anda CDR3 as set forth in SEQ ID NO:
 13. 7. The formulation of claim 1,wherein said antibody is vedolizumab.
 8. The formulation of claim 6,wherein said antibody is vedolizumab.
 9. The formulation of claim 1,wherein the anti-α4β7 antibody comprises a heavy chain variable regioncomprising amino acids 20 to 140 of SEQ ID NO: 2 and a light chainvariable region comprising amino acids 20 to 131 of SEQ ID NO:
 4. 10.The formulation of claim 1, wherein the formulation is in a vial, andthe vial contains about 300 mg of the anti-α4β7 antibody.
 11. Theformulation of claim 3, wherein the histidine amount is 3% to 6% (w/w ofdry formulation).
 12. The formulation of claim 1, wherein theformulation is stable at 25° C. and 60% relative humidity (RH) for atleast 12 months.
 13. A stable pharmaceutical formulation comprising 45%to 55% sucrose (w/w of dry formulation), at least about 240 mg of ahumanized anti-α4β7 antibody, histidine, about 10% to about 15% w/w ofarginine, and polysorbate 80, wherein the formulation is a lyophilizedformulation, wherein the formulation comprises at least 50% majorisoform of the antibody as determined by Cation Exchange Chromatography(CEX), and wherein the antibody comprises a light chain variable regioncomprising amino acids 20-131 of SEQ ID NO: 4, and a heavy chainvariable region comprising amino acids 20 to 140 of SEQ ID NO:
 2. 14.The formulation of claim 13, wherein the antibody is vedolizumab. 15.The formulation of claim 13, wherein the sucrose to antibody ratio is atleast 700:1.
 16. The formulation of claim 6, comprising 300 mg of theantibody.
 17. A liquid, reconstituted formulation comprising about 50mg/ml to about 80 mg/ml of an anti-α4β7 antibody, about 250 mM to about325 mM sucrose, about 75 mM to about 200 mM arginine, about 25 mM toabout 150 mM histidine, and about 0.01% to about 0.1% polysorbate 80,wherein the formulation comprises at least 50% major isoform of theantibody as determined by Cation Exchange Chromatography (CEX), whereinthe antibody is an IgG1 and comprises a light chain variable regioncomprising amino acids 20-131 of SEQ ID NO: 4 and a heavy chain variableregion comprising amino acids 20 to 140 of SEQ ID NO: 2, and wherein theformulation has a pH between about 5.5 to 6.5.
 18. The formulation ofclaim 17, having a pH between about 6.1 to 6.5.
 19. The formulation ofclaim 17, wherein the antibody is vedolizumab.
 20. The formulation ofclaim 19, comprising 300 mg of vedolizumab.
 21. The formulation of claim17, comprising about 25 mM to about 65 mM histidine.
 22. The formulationof claim 17, comprising about 100 mM to about 150 mM arginine.
 23. Theformulation of claim 1, comprising greater than about 13% arginine (w/wof the lyophilized formulation) when the protein amount is about 31%(w/w of the lyophilized formulation).
 24. The formulation of claim 6,comprising greater than about 13% arginine (w/w of the lyophilizedformulation) when the protein amount is about 31% (w/w of thelyophilized formulation).
 25. The formulation of claim 17, comprisinggreater than about 13% arginine (w/w of the lyophilized formulation)when the protein amount is about 31% (w/w of the lyophilizedformulation).
 26. The formulation of claim 6, wherein the anti-α4β7antibody comprises a heavy chain variable region comprising amino acids20 to 140 of SEQ ID NO: 2 and a light chain variable region comprisingamino acids 20 to 131 of SEQ ID NO:
 4. 27. The formulation of claim 1,wherein said formulation comprises at least about 25% anti-α4β7 antibody(by weight).
 28. The formulation of claim 1, comprising at least 55%major isoform of the antibody as determined by CEX.
 29. The formulationof claim 6, wherein the formulation has a pH of about 5.5 to 6.5 uponreconstitution.
 30. The formulation of claim 2, wherein the formulationhas a pH of about 5.5 to 6.5 upon reconstitution.
 31. The formulation ofclaim 1, wherein the antibody is an IgG1.
 32. The formulation of claim6, wherein the antibody is an IgG1.
 33. The formulation of claim 13,wherein the antibody is an IgG1.